Methods for rapid forensic analysis of mitochondrial DNA

ABSTRACT

The present invention provides methods for rapid forensic analysis of mitochondrial DNA by amplification of a segment of mitochondrial DNA containing restriction sites, digesting the mitochondrial DNA segments with restriction enzymes, determining the molecular masses of the restriction fragments and comparing the molecular masses with the molecular masses of theoretical restriction digests of known mitochondrial DNA sequences stored in a database.

FIELD OF THE INVENTION

This invention relates to the field of mitochondrial DNA analysis. Theinvention enables rapid and accurate forensic analysis by using massspectrometry to characterize informative regions of mitochondrial DNA.

BACKGROUND OF THE INVENTION

Mitochondrial DNA (mtDNA) is found in eukaryotes and differs fromnuclear DNA in its location, its sequence, its quantity in the cell, andits mode of inheritance. The nucleus of the cell contains two sets of 23chromosomes, one paternal set and one maternal set. However, cells maycontain hundreds to thousands of mitochondria, each of which may containseveral copies of mtDNA. Nuclear DNA has many more bases than mtDNA, butmtDNA is present in many more copies than nuclear DNA. Thischaracteristic of mtDNA is useful in situations where the amount of DNAin a sample is very limited. Typical sources of DNA recovered from crimescenes include hair, bones, teeth, and body fluids such as saliva,semen, and blood.

In humans, mitochondrial DNA is inherited strictly from the mother (CaseJ. T. and Wallace, D. C., Somatic Cell Genetics, 1981, 7, 103-108;Giles, R. E. et al. Proc. Natl. Acad. Sci. 1980, 77, 6715-6719;Hutchison, C. A. et al. Nature, 1974, 251, 536-538). Thus, the mtDNAsequences obtained from maternally related individuals, such as abrother and a sister or a mother and a daughter, will exactly match eachother in the absence of a mutation. This characteristic of mtDNA isadvantageous in missing persons cases as reference mtDNA samples can besupplied by any maternal relative of the missing individual (Ginther, C.et al. Nature Genetics, 1992, 2, 135-138; Holland, M. M. et al. Journalof Forensic Sciences, 1993, 38, 542-553; Stoneking, M. et al. AmericanJournal of Human Genetics, 1991, 48, 370-382).

The human mtDNA genome is approximately 16,569 bases in length and hastwo general regions: the coding region and the control region. Thecoding region is responsible for the production of various biologicalmolecules involved in the process of energy production in the cell andincludes about 37 genes (22 transfer RNAs, 2 ribosomal RNAs, and 13peptides), with very little intergenic sequence and no introns. Thecontrol region is responsible for regulation of the mtDNA molecule. Tworegions of mtDNA within the control region have been found to be highlypolymorphic, or variable, within the human population (Greenberg, B. D.et al. Gene, 1983, 21, 33-49). These two regions are termed“hypervariable Region I” (HV1), which has an approximate length of 342base pairs (bp), and “hypervariable Region II” (HV2), which has anapproximate length of 268 bp. Forensic mtDNA examinations are performedusing these two regions because of the high degree of variability foundamong individuals.

There exists a need for rapid identification of humans wherein humanremains and/or biological samples are analyzed. Such remains or samplesmay be associated with war-related casualties, aircraft crashes, andacts of terrorism, for example. Analysis of mtDNA enables arule-in/rule-out identification process for persons for whom DNAprofiles from a maternal relative are available. Human identification byanalysis of mtDNA can also be applied to human remains and/or biologicalsamples obtained from crime scenes.

The process of human identification is a common objective of forensicsinvestigations. As used herein, “forensics” is the study of evidencediscovered at a crime or accident scene and used in a court of law.“Forensic science” is any science used for the purposes of the law, inparticular the criminal justice system, and therefore provides impartialscientific evidence for use in the courts of law, and in a criminalinvestigation and trial. Forensic science is a multidisciplinarysubject, drawing principally from chemistry and biology, but also fromphysics, geology, psychology and social science, for example.

Forensic scientists generally use two highly variable regions of humanmtDNA for analysis. These regions are designated “hypervariable regions1 and 2” (HV1 and HV2 which contain 341 and 267 base pairsrespectively). These hypervariable regions, or portions thereof, provideone non-limiting example of mitochondrial DNA identifying amplicons.

A typical mtDNA analysis begins when total genomic DNA is extracted frombiological material, such as a tooth, blood sample, or hair. Thepolymerase chain reaction (PCR) is then used to amplify, or create manycopies of, the two hypervariable portions of the non-coding region ofthe mtDNA molecule, using flanking primers. Care is taken to eliminatethe introduction of exogenous DNA during both the extraction andamplification steps via methods such as the use of pre-packaged sterileequipment and reagents, aerosol-resistant barrier pipette tips, gloves,masks, and lab coats, separation of pre- and post-amplification areas inthe lab using dedicated reagents for each, ultraviolet irradiation ofequipment, and autoclaving of tubes and reagent stocks. In casework,questioned samples are always processed before known samples and theyare processed in different laboratory rooms. When adequate amounts ofPCR product are amplified to provide all the necessary information aboutthe two hypervariable regions, sequencing reactions are performed. Thesechemical reactions use each PCR product as a template to create a newcomplementary strand of DNA in which some of the nucleotide residuesthat make up the DNA sequence are labeled with dye. The strands createdin this stage are then separated according to size by an automatedsequencing machine that uses a laser to “read” the sequence, or order,of the nucleotide bases. Where possible, the sequences of bothhypervariable regions are determined on both strands of thedouble-stranded DNA molecule, with sufficient redundancy to confirm thenucleotide substitutions that characterize that particular sample. Atleast two forensic analysts independently assemble the sequence and thencompare it to a standard, commonly used, reference sequence. The entireprocess is then repeated with a known sample, such as blood or salivacollected from a known individual. The sequences from both samples,about 780 bases long each, are compared to determine if they match. Theanalysts assess the results of the analysis and determine if anyportions of it need to be repeated. Finally, in the event of aninclusion or match, the SWGDAM mtDNA database, which is maintained bythe FBI, is searched for the mitochondrial sequence that has beenobserved for the samples. The analysts can then report the number ofobservations of this type based on the nucleotide positions that havebeen read. A written report can be provided to the submitting agency.

Approximately 610 bp of mtDNA are currently sequenced in forensic mtDNAanalysis. Recording and comparing mtDNA sequences would be difficult andpotentially confusing if all of the bases were listed. Thus, mtDNAsequence information is recorded by listing only the differences withrespect to a reference DNA sequence. By convention, human mtDNAsequences are described using the first complete published mtDNAsequence as a reference (Anderson, S. et al., Nature, 1981, 290,457-465). This sequence is commonly referred to as the Andersonsequence. It is also called the Cambridge reference sequence or theOxford sequence. Each base pair in this sequence is assigned a number.Deviations from this reference sequence are recorded as the number ofthe position demonstrating a difference and a letter designation of thedifferent base. For example, a transition from A to G at Position 263would be recorded as 263 G. If deletions or insertions of bases arepresent in the mtDNA, these differences are denoted as well.

In the United States, there are seven laboratories currently conductingforensic mtDNA examinations: the FBI Laboratory; Laboratory Corporationof America (LabCorp) in Research Triangle Park, North Carolina;Mitotyping Technologies in State College, Pennsylvania; the BodeTechnology Group (BTG) in Springfield, Virginia; the Armed Forces DNAIdentification Laboratory (AFDIL) in Rockville, Md.; BioSynthesis, Inc.in Lewisville, Texas; and Reliagene in New Orleans, La.

Mitochondrial DNA analyses have been admitted in criminal proceedingsfrom these laboratories in the following states as of April 1999:Alabama, Arkansas, Florida, Indiana, Illinois, Maryland, Michigan, NewMexico, North Carolina, Pennsylvania, South Carolina, Tennessee, Texas,and Washington. Mitochondrial DNA has also been admitted and used incriminal trials in Australia, the United Kingdom, and several otherEuropean countries.

Since 1996, the number of individuals performing mitochondrial DNAanalysis at the FBI Laboratory has grown from 4 to 12, with morepersonnel expected in the near future. Over 150 mitochondrial DNA caseshave been completed by the FBI Laboratory as of March 1999, and dozensmore await analysis. Forensic courses are being taught by the FBILaboratory personnel and other groups to educate forensic scientists inthe procedures and interpretation of mtDNA sequencing. More and moreindividuals are learning about the value of mtDNA sequencing forobtaining useful information from evidentiary samples that are small,degraded, or both. Mitochondrial DNA sequencing is becoming known notonly as an exclusionary tool but also as a complementary technique foruse with other human identification procedures. Mitochondrial DNAanalysis will continue to be a powerful tool for law enforcementofficials in the years to come as other applications are developed,validated, and applied to forensic evidence.

Presently, the forensic analysis of mtDNA is rigorous andlabor-intensive. Currently, only 1-2 cases per month per analyst can beperformed. Several molecular biological techniques are combined toobtain a mtDNA sequence from a sample. The steps of the mtDNA analysisprocess include primary visual analysis, sample preparation, DNAextraction, polymerase chain reaction (PCR) amplification,post-amplification quantification of the DNA, automated DNA sequencing,and data analysis. Another complicating factor in the forensic analysisof mtDNA is the occurrence of heteroplasmy wherein the pool of mtDNAs ina given cell is heterogeneous due to mutations in individual mtDNAs.There are two forms of heteroplasmy found in mtDNA. Sequenceheteroplasmy (also known as point heteroplasmy) is the occurrence ofmore than one base at a particular position or positions in the mtDNAsequence. Length heteroplasmy is the occurrence of more than one lengthof a stretch of the same base in a mtDNA sequence as a result ofinsertion of nucleotide residues.

Heteroplasmy is a problem for forensic investigators since a sample froma crime scene can differ from a sample from a suspect by one base pairand this difference may be interpreted as sufficient evidence toeliminate that individual as the suspect. Hair samples from a singleindividual can contain heteroplasmic mutations at vastly differentconcentrations and even the root and shaft of a single hair can differ.The detection methods currently available to molecular biologists cannotdetect low levels of heteroplasmy. Furthermore, if present, lengthheteroplasmy will adversely affect sequencing runs by resulting in anout-of-frame sequence that cannot be interpreted.

Mass spectrometry provides detailed information about the moleculesbeing analyzed, including high mass accuracy. It is also a process thatcan be easily automated.

Several groups have described detection of PCR products using highresolution electrospray ionization-Fourier transform-ion cyclotronresonance mass spectrometry (ESI-FT-ICR MS). Accurate measurement ofexact mass combined with knowledge of the number of at least onenucleotide allowed calculation of the total base composition for PCRduplex products of approximately 100 base pairs. (Aaserud et al., J. Am.Soc. Mass Spec., 1996, 7, 1266-1269; Muddiman et al., Anal. Chem., 1997,69, 1543-1549; Wunschel et al., Anal. Chem., 1998, 70, 1203-1207;Muddiman et al., Rev. Anal. Chem., 1998, 17, 1-68). Electrosprayionization-Fourier transform-ion cyclotron resistance (ESI-FT-ICR) MSmay be used to determine the mass of double-stranded, 500 base-pair PCRproducts via the average molecular mass (Hurst et al., Rapid Commun.Mass Spec. 1996, 10, 377-382). The use of matrix-assisted laserdesorption ionization-time of flight (MALDI-TOF) mass spectrometry forcharacterization of PCR products has been described. (Muddiman et al.,Rapid Commun. Mass Spec., 1999, 13, 1201-1204). However, the degradationof DNAs over about 75 nucleotides observed with MALDI limited theutility of this method.

U.S. Pat. No. 5,849,492 reports a method for retrieval ofphylogenetically informative DNA sequences which comprise searching fora highly divergent segment of genomic DNA surrounded by two highlyconserved segments, designing the universal primers for PCRamplification of the highly divergent region, amplifying the genomic DNAby PCR technique using universal primers, and then sequencing the geneto determine the identity of the organism.

U.S. Pat. No. 5,965,363 reports methods for screening nucleic acids forpolymorphisms by analyzing amplified target nucleic acids using massspectrometric techniques and to procedures for improving mass resolutionand mass accuracy of these methods.

WO 99/14375 reports methods, PCR primers and kits for use in analyzingpreselected DNA tandem nucleotide repeat alleles by mass spectrometry.

WO 98/12355 reports methods of determining the mass of a target nucleicacid by mass spectrometric analysis, by cleaving the target nucleic acidto reduce its length, making the target single-stranded and using MS todetermine the mass of the single-stranded shortened target. Alsoreported are methods of preparing a double-stranded target nucleic acidfor MS analysis comprising amplification of the target nucleic acid,binding one of the strands to a solid support, releasing the secondstrand and then releasing the first strand which is then analyzed by MS.Kits for target nucleic acid preparation are also reported.

PCT WO97/33000 reports methods for detecting mutations in a targetnucleic acid by nonrandomly fragmenting the target into a set ofsingle-stranded nonrandom length fragments and determining their massesby MS.

U.S. Pat. No. 5,605,798 reports a fast and highly accurate massspectrometer-based process for detecting the presence of a particularnucleic acid in a biological sample for diagnostic purposes.

WO 98/20166 reports processes for determining the sequence of aparticular target nucleic acid by mass spectrometry. Processes fordetecting a target nucleic acid present in a biological sample by PCRamplification and mass spectrometry detection are disclosed, as aremethods for detecting a target nucleic acid in a sample by amplifyingthe target with primers that contain restriction sites and tags,extending and cleaving the amplified nucleic acid, and detecting thepresence of extended product, wherein the presence of a DNA fragment ofa mass different from wild-type is indicative of a mutation. Methods ofsequencing a nucleic acid via mass spectrometry methods are alsodescribed.

WO 97/37041, WO 99/31278 and U.S. Pat. No. 5,547,835 report methods ofsequencing nucleic acids using mass spectrometry. U.S. Pat. Nos.5,622,824, 5,872,003 and 5,691,141 report methods, systems and kits forexonuclease-mediated mass spectrometric sequencing.

There is a need for a mitochondrial DNA forensic analysis which is bothspecific and rapid, and in which no nucleic acid sequencing is required.The present invention addresses this need, among others.

SUMMARY OF THE INVENTION

The present invention is directed to methods of forensic analysis ofmitochondrial DNA comprising: amplifying a segment of mitochondrial DNAcontaining a plurality of restriction sites and flanked by a pair ofprimers to produce an amplification product, digesting the amplificationproduct with a plurality of restriction enzymes to produce a pluralityof restriction digest products, determining the molecular mass of eachmember of the plurality of restriction digest products, generating afragment coverage map from the molecular masses and comparing thefragment coverage map with a plurality of theoretical fragment coveragemaps contained in a database stored on a computer readable medium.

The present invention is also directed to primer pair compositions usedto amplify mitochondrial DNA for the forensic method and to isolatedmitochondrial DNA amplicons obtained by amplification of mitochondrialDNA with the primer pair compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of base composition determination usingnucleotide analog “tags” to determine base compositions.

FIG. 2 shows the deconvoluted mass spectra of a Bacillus anthracisregion with and without the mass tag phosphorothioate A (A*). The twospectra differ in that the measured molecular weight of the masstag-containing sequence is greater than that of the unmodified sequence.

FIG. 3 indicates the process of mtDNA analysis. After amplification byPCR (210), the PCR products were subjected to restriction digests (220)with RsaI for HV1 and a combination of HpaII, HpyCH41V, PacI and EaeIfor HV2 in order to obtain amplicon segments suitable for analysis byFTICR-MS (240). The data were processed to obtain mass data for eachamplicon segment (250) which were then compared to the masses calculatedfor theoretical digests from the FBI mtDNA database by a scoring scheme(260).

FIG. 4 is a comparison of two mass spectra which indicates that the useof exo(−) pfu polymerase prevents addition of non-templated adenosineresidues and results in a strong signal, relative to the use of thecommonly used Amplitaq™ gold polymerase.

FIG. 5 indicates that gel electrophoresis confirms that exo(−) pfupolymerase is equally effective as a standard polymerase inamplification of mtDNA obtained from blood, fingernail and salivasamples.

FIG. 6 exhibits two plots that indicate positions of cleavage of humanmtDNA obtained with different panels of restriction endonucleases. Themodified panel wherein EaeI and PacI are replaced with HaeIII andHpyCH4IV respectively, results in better spacing of conservedrestriction sites.

FIG. 7 is an agarose gel electrophoresis photo confirming the activityof restriction endonucleases: EaeI, HpyCH4IV, HpyCH4IV, HpaII, PacI andHaeIII on Hv2 amplicon from a mtDNA preparation obtained from a bloodsample (Seracare N31773).

FIG. 8 is an agarose gel electrophoresis photo confirming that theprimers designed to amplify the 12 non-control regions (Regions R1-R12)produce amplicons of the expected sizes.

FIG. 9 is an agarose gel electrophoresis photo indicating thesensitivity of the Hv1 and Hv2 primer pairs assessed against DNAisolated from human blood. A PCR product is detectable down to between160 pg and 1.6 ng for both HV1 and HV2 primer pairs.

FIG. 10 is an agarose gel electrophoresis photo indicating that PCRproducts are obtained for each of the 36 samples described in Example 13when amplified with HV1 primers.

DESCRIPTION OF EMBODIMENTS

The present invention provides, inter alia, methods for forensicanalysis of mitochondrial DNA. A region of mitochondrial DNA whichcontains on or more restriction sites is selected to provide optimaldistinguishing capability which enables forensic conclusions to bedrawn. A relational database of known mitochondrial DNA sequences isthen populated with the results of theoretical restriction digestionreactions. One or more primer pairs are then selected to amplify theregion of mitochondrial DNA and amplification product is digested withone or more restriction enzymes which are chosen to yield restrictionfragments of up to about 150 base pairs that are amenable to molecularmass analysis. The molecular masses of all of the restriction fragmentsare then measured and the results are compared with the resultscalculated for the theoretical restriction digestions of all of theentries in the relational database. The results of the comparison enablea forensic conclusion to be drawn.

In one embodiment, more than one region can be analyzed to draw aforensic conclusion via a triangulation strategy. For example, it ispossible that analysis of one region of DNA obtained from a crime sceneyields several possible matches to entries in a relational database. Inthis case, depending on the objective of the individual forensicanalysis, it may be advantageous to carry out one or more additionalanalyses of different mtDNA regions. Examples of such mtDNA regionsinclude, but are not limited to a portion of, HV1, HV2, R1, R2, R3, R4,R5, R6, R7, R8, R9, R10, R1 and R12 (coordinates for each of thesedefined regions, relative to the Anderson Sequence are given in Table2). Thus, in this embodiment, any combination of two or more regions ofmtDNA are used to provide optimal distinguishing capability and providean improved confidence level for the forensic analysis.

In another embodiment, the relational database of known mitochondrialDNA sequences is populated with base compositions of the theoreticalrestriction fragments obtained from theoretical digestion of each memberof the database. Then the base compositions of each of the restrictionfragments of the experimentally determined molecular masses aredetermined. The analysis may then end with a comparison of theexperimentally determined base compositions with the base compositionsof the theoretical digestions of each member of the database so that atleast one base composition match or lack of a base composition matchprovides a forensic conclusion.

In another embodiment, one or more restriction enzymes which are chosento yield restriction fragments of up to about 50 base pairs, of up toabout 100 base pairs, of up to about 150 base pairs, of up to about 200base pairs, or of up to about 250 base pairs that are amenable tomolecular mass analysis.

In another embodiment, the molecular masses of all or most (i.e., about75%, about 80%, about 90% about 99% or every fragment minus onefragment) of the restriction fragments are then measured and the resultsare compared with the results calculated for the theoretical restrictiondigestions of all of the entries in the relational database.

In some embodiments, the amplifying step is accomplished by using thepolymerase chain reaction and a polymerase chain reaction is catalyzedby a polymerase enzyme whose function is modified relative to a nativepolymerase. In some embodiments the modified polymerase enzyme is exo(−)Pfu polymerase which catalyzes the addition of nucleotide residues tostaggered restriction digest products to convert the staggered digestproducts to blunt-ended digest products.

Although the use of PCR is suitable, other nucleic acid amplificationtechniques may also be used, including ligase chain reaction (LCR) andstrand displacement amplification (SDA).

Mass spectrometry (MS)-based detection of PCR products provides a meansfor determination of BCS which has several advantages. MS isintrinsically a parallel detection scheme without the need forradioactive or fluorescent labels, since every amplification product isidentified by its molecular mass. The current state of the art in massspectrometry is such that less than femtomole quantities of material canbe readily analyzed to afford information about the molecular contentsof the sample. An accurate assessment of the molecular mass of thematerial can be quickly obtained, irrespective of whether the molecularweight of the sample is several hundred, or in excess of one hundredthousand atomic mass units (amu) or Daltons. Intact molecular ions canbe generated from amplification products using one of a variety ofionization techniques to convert the sample to gas phase. Theseionization methods include, but are not limited to, electrosprayionization (ES), matrix-assisted laser desorption ionization (MALDI) andfast atom bombardment (FAB). For example, MALDI of nucleic acids, alongwith examples of matrices for use in MALDI of nucleic acids, aredescribed in WO 98/54751. The accurate measurement of molecular mass forlarge DNAs is limited by the adduction of cations from the PCR reactionto each strand, resolution of the isotopic peaks from natural abundance¹³C and ¹⁵N isotopes, and assignment of the charge state for any ion.The cations are removed by in-line dialysis using a flow-through chipthat brings the solution containing the PCR products into contact with asolution containing ammonium acetate in the presence of an electricfield gradient orthogonal to the flow. The latter two problems areaddressed by operating with a resolving power of >100,000 and byincorporating isotopically depleted nucleotide triphosphates into theDNA. The resolving power of the instrument is also a consideration. At aresolving power of 10,000, the modeled signal from the [M-14H+]¹⁴⁻charge state of an 84mer PCR product is poorly characterized andassignment of the charge state or exact mass is impossible. At aresolving power of 33,000, the peaks from the individual isotopiccomponents are visible. At a resolving power of 100,000, the isotopicpeaks are resolved to the baseline and assignment of the charge statefor the ion is straightforward. The [¹³C, ¹⁵N]-depleted triphosphatesare obtained, for example, by growing microorganisms on depleted mediaand harvesting the nucleotides (Batey et al., Nucl. Acids Res., 1992,20, 4515-4523).

While mass measurements of intact nucleic acid regions are believed tobe adequate, tandem mass spectrometry (MS^(n)) techniques may providemore definitive information pertaining to molecular identity orsequence. Tandem MS involves the coupled use of two or more stages ofmass analysis where both the separation and detection steps are based onmass spectrometry. The first stage is used to select an ion or componentof a sample from which further structural information is to be obtained.The selected ion is then fragmented using, e.g., blackbody irradiation,infrared multiphoton dissociation, or collisional activation. Forexample, ions generated by electrospray ionization (ESI) can befragmented using IR multiphoton dissociation. This activation leads todissociation of glycosidic bonds and the phosphate backbone, producingtwo series of fragment ions, called the w-series (having an intact 3′terminus and a 5′ phosphate following internal cleavage) and the α-Baseseries (having an intact 5′ terminus and a 3′ furan).

The second stage of mass analysis is then used to detect and measure themass of these resulting fragments of product ions. Such ion selectionfollowed by fragmentation routines can be performed multiple times so asto essentially completely dissect the molecular sequence of a sample.

If there are two or more targets of similar molecular mass, or if asingle amplification reaction results in a product which has the samemass as two or more reference standards, they can be distinguished byusing mass-modifying “tags.” In this embodiment of the invention, anucleotide analog or “tag” is incorporated during amplification (e.g., a5-(trifluoromethyl) deoxythymidine triphosphate) which has a differentmolecular weight than the unmodified base so as to improve distinctionof masses. Such tags are described in, for example, PCT WO97/33000,which is incorporated herein by reference in its entirety. This furtherlimits the number of possible base compositions consistent with anymass. For example, 5-(trifluoromethyl)deoxythymidine triphosphate can beused in place of dTTP in a separate nucleic acid amplification reaction.Measurement of the mass shift between a conventional amplificationproduct and the tagged product is used to quantitate the number ofthymidine nucleotides in each of the single strands. Because the strandsare complementary, the number of adenosine nucleotides in each strand isalso determined.

In another amplification reaction, the number of G and C residues ineach strand is determined using, for example, the cytidine analog5-methylcytosine (5-meC) or 5prolynylcytosine propyne C. The combinationof the A/T reaction and G/C reaction, followed by molecular weightdetermination, provides a unique base composition. This method issummarized in FIG. 1 and Table 1. TABLE 1 Total Total Total Base Basebase base mass info info comp. comp. Double strand Single strand thisthis other Top Bottom Mass tag sequence Sequence strand strand strandstrand strand T*.mass T*ACGT*ACGT* T*ACGT*ACGT* 3x 3T 3A 3T 3A (T* − T)= x AT*GCAT*GCA 2A 2T 2C 2G 2G 2C AT*GCAT*GCA 2x 2T 2A C*.massTAC*GTAC*GT TAC*GTAC*GT 2x 2C 2G (C* − C) = y ATGC*ATGC*A ATGC*ATGC*A 2x2C 2G

The mass tag phosphorothioate A (A*) was used to distinguish a Bacillusanthracis cluster. The B. anthracis (A₁₄G₉C₁₄T₉) had an average MW of14072.26, and the B. anthracis (A₁A*₁₃G₉C₁₄T₉) had an average molecularweight of 14281.11 and the phosphorothioate A had an average molecularweight of +16.06 as determined by ESI-TOF MS. The deconvoluted spectraare shown in FIG. 2.

In another example, assume the measured molecular masses of each strandare 30,000.115Da and 31,000.115 Da respectively, and the measured numberof dT and dA residues are (30,28) and (28,30). If the molecular mass isaccurate to 100 ppm, there are 7 possible combinations of dG+dC possiblefor each strand. However, if the measured molecular mass is accurate to10 ppm, there are only 2 combinations of dG+dC, and at 1 ppm accuracythere is only one possible base composition for each strand.

Signals from the mass spectrometer may be input to a maximum-likelihooddetection and classification algorithm such as is widely used in radarsignal processing. Processing may end with a Bayesian classifier usinglog likelihood ratios developed from the observed signals and averagebackground levels. Background signal strengths are estimated and usedalong with the matched filters to form signatures which are thensubtracted the maximum likelihood process is applied to this “cleanedup” data in a similar manner employing matched filters and a running-sumestimate of the noise-covariance for the cleaned up data.

In some embodiments, the mitochondrial DNA analyzed is humanmitochondrial DNA obtained from human saliva, hair, blood, or nail. Inother embodiments, the DNA analyzed can be obtained from an animal, afungus, a parasite or a protozoan.

The present invention also comprises primer pairs which are designed tobind to highly conserved sequence regions mitochondrial DNA that flankan intervening variable region such as the variable sections foundwithin regions HV1, HV2, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11and R12 and yield amplification products which ideally provide enoughvariability to provide a forensic conclusion, and which are amenable tomolecular mass analysis. By the term “highly conserved,” it is meantthat the sequence regions exhibit from about 80 to 100%, or from about90 to 100%, or from about 95 to 100% identity, or from about 80 to 99%,or from about 90 to 99%, or from about 95 to 99% identity. The molecularmass of a given amplification product provides a means of drawing aforensic conclusion due to the variability of the variable region. Thus,design of primers involves selection of a variable section with optimalvariability in the mtDNA of different individuals.

In some embodiments, each member of the pair has at least 70%, at least80%, at least 90%, at least 95%, or at least 99% sequence identity withthe sequence of the corresponding member of any one or more of thefollowing primer pair sequences: SEQ ID NOs: 8:9, 10:11, 12:13, 12:14,12:15, 16:17, 18:19, 20:21, 22:23, 24:25, 26:27, 28:29, 30:31, 32:33,34:35, 36:37, 38:39, 40:41, 42:43, 44:45, 42:46, 47:48, 18:49, 50:51,22:52, 53:54, 55:56, 57:29, 58:31, 59:60, 61:62, 63:39, 40:64, 65:66,67:68, 69:70, 12:68, 12:70, 67:15, 71:70, 69:15, and 69:68.

In some embodiments, the region of mitochondrial DNA comprises HV1, eachmember of the primer pair has at least 70% sequence identity with thesequence of the corresponding member of any one of the following primerpair sequences: SEQ ID NOs: 12:13, 12:14, 12:15, 16: 17, 42:43, 42:46,67:68, 69:70, 12:68, 12:70, 67:15, 71:70, 69:15, or 69:68, and therestriction enzyme is RsaI.

In some embodiments, the region of mitochondrial DNA comprises HV2, eachmember of the primer pair has at least 70% sequence identity with thesequence of the corresponding member of any one of the following primerpair sequences: SEQ ID NOs: 8:9, 10:11, 16:17, or 65:66, and the atleast one restriction enzyme is HaeIII, HpaII, MfeI, or SspI, or HpaII,HpyCH4IV, PacI, or EaeI.

In some embodiments, the region of mitochondrial DNA comprises regionR1, each member of the primer pair has at least 70% sequence identitywith the sequence of the corresponding member of any one of thefollowing primer pair sequences: SEQ ID NOs: 18:19 and 18:49, at leastone restriction enzyme is DdeI, MseI, HaeIII, or MboI.

In some embodiments, the region of mitochondrial DNA comprises regionR2, each member of the primer pair has at least 70% sequence identitywith the sequence of the corresponding member of any one of thefollowing primer pair sequences: SEQ ID NOs: 20:21 and 50:51, and atleast one restriction enzyme is DdeI, HaeIII, MboI, or MseI.

In some embodiments, the region of mitochondrial DNA comprises regionR3, each member of the primer pair has at least 70% sequence identitywith the sequence of the corresponding member of any one of thefollowing primer pair sequences: SEQ ID NOs: 22:23 and 22:52, and atleast one restriction enzyme is DdeI, MseI, MboI, or BanI.

In some embodiments, the region of mitochondrial DNA comprises regionR4, each member of the primer pair has at least 70% sequence identitywith the sequence of the corresponding member of any one of thefollowing primer pair sequences: SEQ ID NOs: 24:25 and 53:54, and atleast one restriction enzyme is DdeI, HpyCH41V, MseI, or HaeIII.

In some embodiments, the region of mitochondrial DNA comprises regionR5, each member of said primer pair has at least 70% sequence identitywith the sequence of the corresponding member of any one of thefollowing primer pair sequences: SEQ ID NOs: 26:27 and 55:56, and atleast one restriction enzyme is AluI, BfaI, or MseI.

In some embodiments, the region of mitochondrial DNA comprises regionR6, each member of the primer pair has at least 70% sequence identitywith the sequence of the corresponding member of any one of thefollowing primer pair sequences: SEQ ID NOs: 28:29 and 57:29, and atleast one restriction enzyme is DdeI, HaeIII, MboI, MseI, or RsaI.

In some embodiments, the region of mitochondrial DNA comprises regionR7, each member of said primer pair has at least 70% sequence identitywith the sequence of the corresponding member of any one of thefollowing primer pair sequences: SEQ ID NOs: 30:31 and 58:31, and atleast one restriction enzyme is DdeI, HpaII, HaeIII, or MseI.

In some embodiments, the region of mitochondrial DNA comprises regionR8, each member of the primer pair has at least 70% sequence identitywith the sequence of the corresponding member of any one of thefollowing primer pair sequences: SEQ ID NOs: 32:33 and 59:60, and atleast one restriction enzyme is BfaI, DdeI, EcoRI, or MboI.

In some embodiments, the region of mitochondrial DNA comprises regionR9, each member of the primer pair has at least 70% sequence identitywith the sequence of the corresponding member of the following primerpair sequences: SEQ ID NOs: 34:35, and at least one restriction enzymeis BfaI, DdeI, HpaII, HpyCH4IV, or MboI.

In some embodiments, the region of mitochondrial DNA comprises regionR10, each member of the primer pair has at least 70% sequence identitywith the sequence of the corresponding member of the following primerpair sequences: SEQ ID NOs: 34:35, and at least one restriction enzymeis BfaI, HpaII, or MboI.

In some embodiments, the region of mitochondrial DNA comprises regionR10, each member of the primer pair has at least 70% sequence identitywith the sequence of the corresponding member of the following primerpair sequences: SEQ ID NOs: 36:37 and 61:62, and at least onerestriction enzyme is BfaI, HpaII, or MboI.

In some embodiments, the region of mitochondrial DNA comprises regionR11, each member of the primer pair has at least 70% sequence identitywith the sequence of the corresponding member of the following primerpair sequences: SEQ ID NOs: 38:39 and 63:39, and at least onerestriction enzyme is BfaI, DdeI, HpyCH4V, or MboI.

In some embodiments, the region of mitochondrial DNA comprises regionR12, each member of the primer pair has at least 70% sequence identitywith the sequence of the corresponding member of the following primerpair sequences: SEQ ID NOs: 40:41 and 40:64, and at least onerestriction enzyme is BfaI, DdeI, or MseI.

Ideally, primer hybridization sites are highly conserved in order tofacilitate the hybridization of the primer. In cases where primerhybridization is less efficient due to lower levels of conservation ofsequence, the primers of the present invention can be chemicallymodified to improve the efficiency of hybridization. For example,because any variation (due to codon wobble in the 3^(rd) position) inthese conserved regions among species is likely to occur in the thirdposition of a DNA triplet, oligonucleotide primers can be designed suchthat the nucleotide corresponding to this position is a base which canbind to more than one nucleotide, referred to herein as a “universalbase.” For example, under this “wobble” pairing, inosine (I) binds to U,C or A; guanine (G) binds to U or C, and uridine (U) binds to U or C.Other examples of universal bases include nitroindoles such as5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides andNucleotides, 1995, 14, 1001-1003), the degenerate nucleotides dP or dK(Hill et al.), an acyclic nucleoside analog containing 5-nitroindazole(Van Aerschot et al., Nucleosides and Nucleotides, 1995, 14, 1053-1056)or the purine analog1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al.,Nucl. Acids Res., 1996, 24, 3302-3306).

In another embodiment of the invention, to compensate for the somewhatweaker binding by the “wobble” base, the oligonucleotide primers aredesigned such that the first and second positions of each triplet areoccupied by nucleotide analogs which bind with greater affinity than theunmodified nucleotide. Examples of these analogs include, but are notlimited to, 2,6-diaminopurine which binds to thymine, propyne T(5-propynyluridine) which binds to adenine and propyne C(5-propynylcytidine) and phenoxazines, including G-clamp, which binds toG. Propynylated pyrimidines are described in U.S. Pat. Nos. 5,645,985,5,830,653 and 5,484,908, each of which is commonly owned andincorporated herein by reference in its entirety. Propynylated primersare claimed in U.S. Ser. No. 10/294,203 which is also commonly owned andincorporated herein by reference in entirety. Phenoxazines are describedin U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096, each of which isincorporated herein by reference in its entirety. G-clamps are describedin U.S. Pat. Nos. 6,007,992 and 6,028,183, each of which is incorporatedherein by reference in its entirety. Thus, In other embodiments, theprimer pair has at least one modified nucleobase such as5-propynylcytidine or 5-propynyluridine.

The present invention also comprises isolated mitochondrial DNAamplicons which are produced by the process of amplification of a sampleof mitochondrial DNA with any of the above-mentioned primers.

While the present invention has been described with specificity inaccordance with certain of its embodiments, the following examples serveonly to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1 Nucleic Acid Isolation and Amplification

General Genomic DNA Sample Prep Protocol: Raw samples were filteredusing Supor-200 0.2 μm membrane syringe filters (VWR International).Samples were transferred to 1.5 ml eppendorf tubes pre-filled with 0.45g of 0.7 mm Zirconia beads followed by the addition of 350 μl of ATLbuffer (Qiagen, Valencia, Calif.). The samples were subjected to beadbeating for 10 minutes at a frequency of 19 l/s in a Retsch VibrationMill (Retsch). After centrifugation, samples were transferred to anS-block plate (Qiagen, Valencia, Calif.) and DNA isolation was completedwith a BioRobot 8000 nucleic acid isolation robot (Qiagen, Valencia,Calif.).

Isolation of Blood DNA—Blood DNA was isolated using an MDx Biorobotaccording to according to the manufacturer's recommended procedure(Isolation of blood DNA on Qiagen QIAamp® DNA Blood BioRobot® MDx Kit,Qiagen, Valencia, Calif.)

Isolation of Buccal Swab DNA—Since the manufacturer does not support afull robotic swab protocol, the blood DNA isolation protocol wasemployed after each swab was first suspended in 400 ml PBS+400 ml QiagenAL buffer+20 μl Qiagen Protease solution in 14 ml round-bottom falcontubes, which were then loaded into the tube holders on the MDx robot.

Isolation of DNA from Nails and Hairs—The following procedure employs aQiagen DNeasy® tissue kit and represents a modification of themanufacturer's suggested procedure: hairs or nails were cut into smallsegments with sterile scissors or razorblades and placed in a centrifugetube to which was added 1 ml of sonication wash buffer (10 mM TRIS-Cl,pH 8.0+10 mM EDTA+0.5% Tween-20. The solution was sonicated for 20minutes to dislodge debris and then washed 2× with 1 ml ultrapure doubledeionized water before addition of 100 μl of Buffer X1 (10 mM TRIS-Cl,ph 8.0+10 mM EDTA+100 mM NaCl+40 mM DTT+2% SDS+250:g/ml Qiagenproteinase K). The sample was then incubated at 55° C. for 1-2 hours,after which 200 μl of Qiagen AL buffer and 210 μl isopropanol were addedand the solution was mixed by vortexing. The sample was then added to aQiagen DNeasy mini spin column placed in a 2 ml collection tube andcentrifuged for 1 min at 6000 g (8000 rpm). Collection tube andflow-through were discarded. The spin column was transferred to a newcollection tube and 500 μl of buffer AW2 was added before centrifugingfor 3 min. at 20,000 g (14,000 rpm) to dry the membrane. For elution,50-100 μl of buffer AE was pipetted directly onto the DNeasy membraneand eluted by centrifugation (6000 g-8000 rpm) after incubation at roomtemperature for 1 min.

Amplification by PCR—An exemplary PCR procedure for amplification ofmitochondrial DNA is the following: A 50 μl total volume reactionmixture contained IX GenAmp® PCR buffer II (Applied Biosystems)-10 mMTRIS-Cl, pH 8.3 and 50 mM KCl, 1.5 mM MgCl₂, 400 mM betaine, 200 μM ofeach dNTP (Stratagene 200415), 250 nM of each primer, and 2.5-5 units ofPfu exo(−) polymerase Gold (Stratagene 600163) and at least 50 pg oftemplate DNA. All PCR solution mixing was performed under aHEPA-filtered positive pressure PCR hood. An example of a programmablePCR cycling profile is as follows: 95° C. for 10 minutes, followed by 8cycles of 95° C. for 20 sec, 62° C. for 20 sec, and 72° C. for 30sec—wherein the 62° C. annealing step is decreased by 1° C. on eachsuccessive cycle of the 8 cycles, followed by 28 cycles of 95° C. for 20sec, 55° C. for 20 sec, and 72° C. for 30 sec, followed by holding at 4°C. Development and optimization of PCR reactions is routine to one withordinary skill in the art and can be accomplished without undueexperimentation.

Example 2 Digestion of Amplicons with Restriction Enzymes

Reaction Conditions—The standard restriction digest reaction conditionsoutlined herein are applicable to all panels of restriction enzymes. ThePCR reaction mixture is diluted into 2×NEB buffer 1+BSA and 1 μl of eachenzyme per 50 μl of reaction mixture is added. The mixture is incubatedat 37° C. for 1 hour followed by 72° C. for 15 minutes. Restrictiondigest enzyme panels for HV1, HV2 and twelve additional regions ofmitochondrial DNA are indicated in Table 2. TABLE 2 mtDNA Regions,Coordinates and Restriction Enzyme Digest Panels COORDINATES RELATIVE TOTHE ANDERSON RESTRICTION mtDNA REGION SEQUENCE (SEQ ID NO: 72) ENZYMEPANEL HV1 (highly variable 16050-16410 RsaI control region 1) HV2(highly variable  29-429 HaeIII HpaII MfeI SspI control region 2) orHpaII, HpyCH4IV, PacI and EaeI REGION R1 (COX2, 8162-8992 DdeI MseIHaeIII MboI Intergenic spacer, tRNA- LYS, ATP6) REGION R2 (ND5)12438-13189 DdeI HaeIII MboI MseI REGION R3 (ND6 tRNA-Glu, 14629-15414DdeI MseI MboI BanI CYTB) REGION R4 (COX3, tRNA- 9435-9461 DdeI HpyCH4IVMseI HaeIII Gly, ND3) REGION R5 (ND4L, ND4) 10753-11500 AluI BfaI MseIREGION R6 (CYTB, tRNA- 15378-16006 DdeI HaeIII MboI MseI RsaI Thr,tRNA-Pro) REGION R7 (ND5, ND6) 13424-14206 DdeI HpaII HaeIII MseI REGIONR8 (ND1) 3452-4210 BfaI DdeI EcoRI MboI REGION R9 (COX2, 7734-8493 BfaIDdeI HpaII HpyCH4IV Intergenic spacer, tRNA- MboI Lys, ATP6) REGION R10(COX1) 6309-7058 BfaI HpaII MboI REGION R11 (COX2, 7644-8371 BfaI DdeIHpyCH4V MboI Intergenic spacer, tRNA- Lys, ATP6) REGION R12 (16S rRNA;2626-3377 BfaI DdeI MseI ND1)

Example 3 Nucleic Acid Purification

Procedure for Semi-Automated Purification of a PCR Mixture UsingCommercially Available Zip Tips®—As Described by Jiang and Hofstadler(Y. Jiang and S. A. Hofstadler Anal. Biochem. 2003, 316, 50-57) anamplified nucleic acid mixture can be purified by commercially availablepipette tips containing anion exchange resin. For pre-treatment ofZipTips® AX (Millipore Corp. Bedford, Mass.), the following steps wereprogrammed to be performed by an Evolution™ P3 liquid handler (PerkinElmer) with fluids being drawn from stock solutions in individual wellsof a 96-well plate (Marshall Bioscience): loading of a rack ofZipTips®AX; washing of ZipTips®AX with 15 μl of 10% NH₄OH/50% methanol;washing of ZipTips® AX with 15 μl of water 8 times; washing of ZipTips®AX with 15 μl of 100 mM NH₄OAc.

For purification of a PCR mixture, 20 μl of crude PCR product wastransferred to individual wells of a MJ Research plate using a BioHit(Helsinki, Finland) multichannel pipette. Individual wells of a 96-wellplate were filled with 300 μl of 40 mM NH₄HCO₃. Individual wells of a96-well plate were filled with 300 μl of 20% methanol. An MJ researchplate was filled with 10 μl of 4% NH₄OH. Two reservoirs were filled withdeionized water. All plates and reservoirs were placed on the deck ofthe Evolution P3 (EP3) (Perkin-Elmer, Boston, Mass.) pipetting stationin pre-arranged order. The following steps were programmed to beperformed by an Evolution P3 pipetting station: aspiration of 20 μl ofair into the EP3 P50 head; loading of a pre-treated rack of ZipTips® AXinto the EP3 P50 head; dispensation of the 20 μl NH₄HCO₃ from theZipTips® AX; loading of the PCR product into the ZipTips® AX byaspiration/dispensation of the PCR solution 18 times; washing of theZipTips® AX containing bound nucleic acids with 15 μl of 40 mM NH₄ HCO₃8 times; washing of the ZipTips® AX containing bound nucleic acids with15 μl of 20% methanol 24 times; elution of the purified nucleic acidsfrom the ZipTips® AX by aspiration/dispensation with 15 μl of 4% NH₄OH18 times. For final preparation for analysis by ESI-MS, each sample wasdiluted 1:1 by volume with 70% methanol containing 50 mM piperidine and50 mM imidazole.

Procedure for Semi-Automated Purification of a PCR mixture with SolutionCapture—The following procedure is disclosed in a U.S. Patentapplication filed on May 12, 2004, (Attorney Docket No. IBIS0026-100):for pre-treatment of ProPac® WAX weak anion exchange resin, thefollowing steps were performed in bulk: sequential washing three times(10:1 volume ratio of buffer to resin) with each of the followingsolutions: (1) 1.0 M formic acid/50% methanol, (2) 20% methanol, (3) 10%NH₄OH, (4) 20% methanol, (5) 40 mM NH₄HCO₃, and (6) 100 mM NH₄OAc. Theresin is stored in 20 mM NH₄OAc/50% methanol at 4° C.

Corning 384-well glass fiber filter plates were pre-treated with tworinses of 250 μl NH₄OH and two rinses of 100 μl NH₄HCO₃.

For binding of the PCR product nucleic acids to the resin, the followingsteps were programmed to be performed by the Evolution™ P3 liquidhandler: addition of 0.05 to 10 μl of pre-treated ProPac® WAX weak anionexchange resin (30 μl of a 1:60 dilution) to a 50 μl PCR reactionmixture (80 μl total volume) in a 96-well plate; mixing of the solutionby aspiration/dispensation for 2.5 minutes; and transfer of the solutionto a pre-treated Corning 384-well glass fiber filter plate. This stepwas followed by centrifugation to remove liquid from the resin and isperformed manually, or under the control of a robotic arm.

The resin containing nucleic acids was then washed by rinsing threetimes with 200 μl of 100 mM NH₄OAc, 200 μl of 40 mM NH₄HCO₃ with removalof buffer by centrifugation for about 15 seconds followed by rinsingthree times with 20% methanol for about 15 seconds. The final rinse wasfollowed by an extended centrifugation step (1-2 minutes).

Elution of the nucleic acids from the resin was accomplished by additionof 40 μl elution/electrospray buffer (25 mM piperidine/25 mMimidazole/35% methanol and 50 nM of an internal standard oligonucleotidefor calibration of mass spectrometry signals) followed by elution fromthe 384-well filter plate into a 384-well catch plate by centrifugation.The eluted nucleic acids in this condition were amenable to analysis byESI-MS. The time required for purification of samples in a single96-well plate using a liquid handler is approximately five minutes.

Example 4 Mass Spectrometry

The mass spectrometer used is a Bruker Daltonics (Billerica, Mass.) ApexII 70e electrospray ionization Fourier transform ion cyclotron resonancemass spectrometer (ESI-FTICR-MS) that employs an actively shielded 7Tesla superconducting magnet. All aspects of pulse sequence control anddata acquisition were performed on a 1.1 GHz Pentium II data stationrunning Bruker's Xmass software. 20 μL sample aliquots were extracteddirectly from 96-well microtiter plates using a CTC HTS PAL autosampler(LEAP Technologies, Carrboro, N.C.) triggered by the data station.Samples were injected directly into the ESI source at a flow rate of 75μL/hr. Ions were formed via electrospray ionization in a modifiedAnalytica (Branford, Conn.) source employing an off axis, groundedelectrospray probe positioned ca. 1.5 cm from the metalized terminus ofa glass desolvation capillary. The atmospheric pressure end of the glasscapillary is biased at 6000 V relative to the ESI needle during dataacquisition. A counter-current flow of dry N₂/O₂ was employed to assistin the desolvation process. Ions were accumulated in an external ionreservoir comprised of an rf-only hexapole, a skimmer cone, and anauxiliary gate electrode, prior to injection into the trapped ion cellwhere they were mass analyzed.

Spectral acquisition was performed in the continuous duty cycle modewhereby ions were accumulated in the hexapole ion reservoirsimultaneously with ion detection in the trapped ion cell. Following a1.2 ms transfer event, in which ions were transferred to the trapped ioncell, the ions were subjected to a 1.6 ms chirp excitation correspondingto 8000-500 m/z. Data was acquired over an m/z range of 500-5000 (IMdata points over a 225 K Hz bandwidth). Each spectrum was the result ofco-adding 32 transients. Transients were zero-filled once prior to themagnitude mode Fourier transform and post calibration using the internalmass standard. The ICR-2LS software package (G. A. Anderson, J. E. Bruce(Pacific Northwest National Laboratory, Richland, Wash., 1995) was usedto deconvolute the mass spectra and calculate the mass of themonoisotopic species using an “averaging” fitting routine (M. W. Senko,S.C. Beu, F. W. McLafferty, J. Am. Soc. Mass Spectrom. 1995, 6, 229)modified for DNA. Using this approach, monoisotopic molecular weightswere calculated.

Example 5 Primer Pairs for Amplification of Informative Regions ofMitochondrial DNA

Conventional forensic mitochondrial DNA analysis typically involvesamplification and sequencing of the two hypervariable regions within thenon-coding control region known as HV1 and HV2. The present inventioncomprises primer pairs for amplification of informative regions withinHV1 and HV2 (SEQ ID NOs: 8-17, 42-48 and 65-71 in Table 3). Additionalindividual discriminating power has been obtained by the selection foranalysis of 12 additional non-control regions (Regions R1-R12) fromwhich informative amplification products of approximately 630-840 bpeach can be obtained using additional primer pairs (SEQ ID NOs: 18-41and 49-70 in Table 3). The primers listed below in Table 3 are generally10-50 nucleotides in length, 15-35 nucleotides in length, or 18-30nucleotides in length.

By convention, human mtDNA sequences are described using the firstcomplete and published mtDNA sequence as a reference (Anderson, S. etal., Nature, 1981, 290, 457-465). This sequence is commonly referred toas the Anderson sequence. Primer pair names on Table 3 indicate themtDNA amplicon coordinates with reference to the Anderson mtDNAsequence: GenBank Accession No. NC_(—)001807.3 (SEQ ID NO: 72). Forexample, primer pairs 8:9 produce an amplicon which corresponds topositions 76-353 of the Anderson sequence. TABLE 3 Primer Pairs forAnalysis of mtDNA FORWARD REVERSE FORWARD SEQ ID REVERSE SEQ ID PRIMERPAIR NAME mtDNA REGION AMPLIFIED PRIMER SEQUENCE NO: PRIMER SEQUENCE NO:HMTHV2_ANDRSN_7 REGION HV2 tcacgcgatagcatt  8 tggtttggcagag  96_353_TMOD gcg atgtgtttaagt HMTHV2_ANDRSN_2 REGION HV2 tctcacgggagctct10 tctgttaaaagtg 11 9_429_TMOD ccatgc cataccgcca HMTHV1_ANDRSN_1 REGIONHV1 tgactcacccatcaa 12 tgaggatggtggt 13 6065_16410_TMOD caaccgc caagggacHMTHV1_ANDRSN_1 REGION HV1 tgactcacccatcaa 12 tggatttgactgt 146065_16354_TMOD caaccgc aatgtgcta HMTHV1_ANDRSN_1 REGION HV1tgactcacccatcaa 12 tgaagggatttga 15 6064_16359 caaccgc ctgtaatgtgcta tgHMT_ASN_16036_5 REGION HV1 and gaagcagatttgggt 16 gtgtgtgtgctgg 17 22REGION HV2 accacc gtaggatg HMT_ASN_8162_89 REGION R1 (COX2,tacggtcaatgctct 18 tggtaagaagtgg 19 16 Intergenic spacer, gaaatctgtgggctagggcatt tRNA-Lys, ATP6) HMT_ASN_12438_1 REGION R2 (ND5)ttatgtaaaatccat 20 tggtgatagcgcc 21 3189 tgtcgcatccacc taagcatagtgHMT_ASN_14629_1 REGION R3 (ND6 tRNA- tcccattactaaacc 22 tttcgtgcaagaa 235353 Glu, CYTB) cacactcaacag taggaggtggag HMT_ASN_9435_10 REGION R4(COX3, taaggccttcgatac 24 tagggtcgaagcc 25 188 tRNA-Gly, ND3)gggataatccta gcactcg HMT_ASN_10753_1 REGION R5 (ND4L, tactccaatgctaaa 26tgtgaggcgtatt 27 1500 ND4) actaatcgtcccaac ataccatagccg HMT_ASN_15369_1REGION R6 (CYTB, tcctaggaatcacct 28 tagaatcttagct 29 6006 tRNA-Thr,tRNA-Pro) cccattccga ttgggtgctaatg gtg HMT_ASN_13461_1 REGION R7 (ND5,ND6) tggcagcctagcatt 30 tggctgaacattg 31 4206 agcaggaata tttgttggtgtHMT_ASN_3452_42 REGION R8 (ND1) tcgctgacgccataa 32 taagtaatgctag 33 10aactcttcac ggtgagtggtagg aag HMT_ASN_7734_84 REGION R9 (COX2,taactaatactaaca 34 tttatgggctttg 35 93 Intergenic spacer,tctcagacgctcagg gtgagggaggta tRNA-Lys, ATP6) a HMT_ASN_6309_70 REGIONR10 (COX1) tactcccaccctgga 36 tgctcctattgat 37 58 gcctc aggacatagtggaagtg HMT_ASN_7644_83 REGION R11 (COX2, ttatcacctttcatg 38 tggcatttcactg39 71 Intergenic spacer, atcacgccct taaagaggtgttg tRNA-Lys, ATP6) gHMT_ASN_2626_33 REGION R12 (16S tgtatgaatggctcc 40 tcggtaagcatta 41 77rRNA; ND1) acgagggt ggaatgccattgc HMTHV1_ANDRSN_1 REGION HV1gactcacccatcaac 42 gaggatggtggtc 43 6065_16410 aaccgc aagggacHMTHV2_ANDRSN_2 REGION HV2 ctcacgggagctctc 44 ctgttaaaagtgc 45 9_429catgc ataccgcca HMTHV1_ANDRSN_1 REGION HV1 gactcacccatcaac 42ggatttgactgta 46 6065_16354 aaccgc atgtgcta HMTHV2_ANDRSN_7 REGION HV2cacgcgatagcattg 47 ggtttggcagaga 48 6_353 cg tgtgtttaagt HMT_ASN_8162_89REGION R1 (COX2, tacggtcaatgctct 18 tggctattggttg 49 92 Intergenicspacer, gaaatctgtgg aatgagtaggctg tRNA-Lys, ATP6) HMT_ASN_12432_1 REGIONR2 (ND5) tccccattatgtaaa 50 tgacttgaagtgg 51 3262 atccattgtcgcagaaggctacg HMT_ASN_14629_1 REGION R3 (ND6 tRNA- tcccattactaaacc 22taagggtggaagg 52 5414 Glu, CYTB) cacactcaacag tgattttatcgga aHMT_ASN_9411_10 REGION R4 (COX3, tgccaccacacacca 53 tatagggtcgaag 54 190tRNA-Gly, ND3) cctg ccgcactc HMT_ASN_10751_1 REGION R5 (ND4L,tctactccaatgcta 55 tggttgagaatga 56 1514 ND4) aaactaatcgtccc gtgtgaggcgHMT_ASN_15378_1 REGION R6 (CYTB, tcacctcccattccg 57 tagaatcttagct 296006 tRNA-Thr, tRNA-Pro) ataaaatcacct ttgggtgctaatg gtg HMT_ASN_13424_1REGION R7 (ND5, ND6) tcaaaaccatacctc 58 tggctgaacattg 31 4206tcacttcaacctc tttgttggtgt HMT_ASN_3443_42 REGION R8 (ND1)tacaacccttcgctg 59 taagtaatgctag 60 10_2 acgccat ggtgagtggtagg aaHMT_ASN_6278_70 REGION R10 (COX1) ttgaacagtctaccc 61 tgtagtacgatgt 62 06tcccttagc ctagtgatgagtt tgc HMT_ASN_7688_83 REGION R11 (COX2,tgcttcctagtcctg 63 tggcatttcactg 39 71 Intergenic spacer, tatgcccttttcctaaagaggtgttg tRNA-Lys, ATP6) g HMT_ASN_2626_34 REGION R12 (16Stgtatgaatggctcc 40 tggcgtcagcgaa 64 63 rRNA; ND1) acgagggt gggttgtaHMTHV2_ASN_72_3 REGION HV2 tgtgcacgcgatagc 65 tggggtttggcag 66 57 attgcgagatgtgtttaag t HMTHV1_ASN_1605 REGION HV1 tcaagtattgactca 67tcgagaagggatt 68 6_16362 cccatcaacaacc tgactgtaatgtg cta HMTHV1_ASN_1605REGION HV1 taccacccaagtatt 69 tcatggggacgag 70 0_16370 gactcacccatcaagggatttgac HMTHV1_ASN_1606 REGION HV1 tgactcacccatcaa 12 tcgagaagggatt68 4_16362 caaccgc tgactgtaatgtg cta HMTHV1_ASN_1606 REGION HV1tgactcacccatcaa 12 tcatggggacgag 70 4_16370 caaccgc aagggatttgacHMTHV1_ASN_1605 REGION HV1 tcaagtattgactca 67 tgaagggatttga 15 6_16359cccatcaacaacc ctgtaatgtgcta tg HMTHV1_ASN_1605 REGION HV1tcaagtattgactca 71 tcatggggacgag 70 6_16370 cccatcaacaacc aagggatttgacHMTHV1_ASN_1605 REGION HV1 taccacccaagtatt 69 tgaagggatttga 15 0_16359gactcacccatc ctgtaatgtgcta tg HMTHV1_ASN_1605 REGION HV1 taccacccaagtatt69 tcgagaagggatt 68 0_16362 gactcacccatc tgactgtaatgtg cta

Example 6 Analysis of 10 Blinded DNA Samples

Ten different blinded samples of human DNA provided by the FBI weresubjected to rapid mtDNA analysis by the method of the present inventionaccording to the process illustrated in FIG. 3. After amplification ofhuman mtDNA by PCR (210), the PCR products were subjected to restrictiondigestion (220) with RsaI for HV1 and a combination of HpaII, HpyCH4IV,PacI and EaeI for HV2 in order to obtain amplicon segments suitable foranalysis by mass spectrometry (230). The data were processed to obtainmass data for each amplicon fragment (240) from which a “fragmentcoverage map” was generated (an example of a fragment coverage map isshown in FIG. 3—represented as a series of horizontal bars beneath themass spectrum). The fragment coverage map was then compared, using ascoring scheme to fragment coverage maps calculated for theoreticaldigests from mtDNA sequences in the FBI mtDNA database (250).

A group of 10 blinded DNA samples was provided by the FBI. HV1 and HV2primer pairs were selected from a sequence alignment created bytranslating the FBI's forensic mtDNA database back into full sequencesvia comparison to the Anderson reference, then selecting primers withinthe full representation core of the alignment and restriction enzymesthat will cleave the 280 and 292 bp PCR products into massspectrometry-compatible fragments. Primer pairs selected foramplification of HV1 segments were SEQ ID NOs: 12:14 and 42:43. Primerpairs selected for amplification of HV2 segments were SEQ ID NOs: 8:9and 44:45 (Table 3). PCR amplification was carried out as indicated inExample 1, with the exception that 2 mM MgCl₂ was included instead of1.5 mM MgCl₂, and that 4 units of Amplitaq Gold® polymerase (AppliedBiosystems) was included instead of 2.5 units of Pfu exo(−) polymerase.3 μl of FBI DNA sample were included in the reaction. Thermal cyclerparameters were as follows: 96° C. for 10 min., followed by 45 cycles ofthe following: 96° C. for 30 sec, 54° C. for 30 sec., and 72° C. for 30sec., after which the reaction was kept at 72° C. for 5 minutes.

Theoretical digestions of the 2754 unambiguous unique sequencescontained within the 4840 FBI sequence entries (there are 399 sequencesin the FBI database which contain at least one ambiguous base callwithin the amplified regions, leading to 4441 unambiguous sequences,2754 of which are unique), with all possible products resulting fromincomplete digestion, were performed and fragment start and endcoordinates, base composition, mass, and end chemistry were stored in adata structure for subsequent fragment pattern reconstruction. Adeconvolved list of monoisotopic exact mass determinations from ICR-2LS₁was determined for each restriction digestion for each blinded sample.For each sample, expected digestion fragment masses were matched toobserved masses with a threshold of ±4 ppm for each database entry (1ppm match error is defined as a difference between observed and expectedmass equal to one millionth of the expected mass).

To evaluate the ability of a single-pass MS-based assay to exclude knowndatabase entries as having base compositions that are different thanthat of an unknown sample, a scoring system was devised that, for agiven input sample, assigns each database sequence a score relative tothe highest scoring sequence. To evaluate whether base composition ofmtDNA fragments can achieve a discrimination power approaching that ofsequencing, the ten blinded samples of human DNA from the FBI wereanalyzed. The overall consistency of the observed digestion productswith the expected fragment pattern for each of the 4840 database entrieswas scored using the sum of four values: 1.) The total number ofobserved masses accounted for in the expected fragment list, 2.) Thepercentage of expected fragments observed for a complete digestion 3.) A“floating percentage” of expected fragments matched, where matches toincomplete digestion fragments were scored ½ percentage point and thetotal number of expected fragments was incremented by ½ for eachobserved incomplete digestion fragment, and 4.) The percentage ofsequence positions accounted for by matches with observed masses. Scoresfor the HV1 and HV2 regions were summed to produce a total score foreach entry. Database entries were sorted by high score and assigned afinal score as a percentage of the top score. An arbitrary (butconservative) scoring threshold of 80% of the top score was set toproduce a very conservative lower bound on the percentage of databaseentries that could be excluded as consistent with each sample.

Without knowing the true sequence of the initial ten samples andallowing for slight experimental variations in restriction digestionsand mass spectrometry, comparison to a large collection of databaseentries enabled exclusion of a vast majority of entries in the database.Table 4 shows an example of the scoring output for one sample (sample 4)and summarizes the exclusion percentages for each of the blinded samplesfor a set of reactions run side-by-side on a single day. The HV1 and HV2regions of each sample were sequenced following the analysis describedin this work for final verification. Table 4 summaries the overallresults of this exercise for this preliminary data analysis. TABLE 4Scoring of FBI Sample 3 Against the FBI Mitochondrial DNA DatabaseNumber of % of % of Floating Database Entry Sequences Sequence FragmentFragment Match Cumulative % Match Row Title Represented Covered CoveredCovered Score Score Score 1 AUT.CAU.000066|USA. 6 99.655 51.04 63.1832.5 333.89 100 CAU.000389|USA.CAU. 000572|USA.CAU.000841| USA.CAU.001074|USA.CAU. 001211 2 USA.CAU.000101 1 90.92 47.02 57.005 24.5300.38 89.9638 3 USA.CAU.000783 1 90.75 44.79 56.37 27 298.08 89.2749 4USA.CAU.000130 1 88.18 46.53 56.68 27.5 296.92 88.9275 5 USA.CAU.0001421 88.18 46.53 56.68 27.5 296.92 88.9275 6 FRA.CAU.000087|GRC. 7 92.76542.71 51.86 25 295.95 88.637 CAU.000032|USA.CAU. 000425|USA.CAU.000483|USA.CAU.000772|USA. CAU.001067|USA.CAU. 001168 44 USA.HIS.000672 1 84.5240.555 46.43 18 268.15 80.3109 45 FRA.CAU.000108|USA. 2 92.055 33.03542.22 17.5 267.68 80.1701 CAU.000890 46 USA.CAU.000361|USA. 2 92.05533.035 42.22 17.5 267.68 80.1701 CAU.001184 47 USA.CAU.001378|USA. 292.055 33.035 42.22 17.5 267.68 80.1701 CAU.001382 48 CHN.ASN.000443 188.525 34.03 43.135 22 267.11 79.9994 49 USA.CAU.000548 1 83.385 39.5847.795 21 266.93 79.9455 50 USA.CAU.000814 1 83.385 39.58 47.795 21266.93 79.9455 51 USA.CAU.000338|USA. 3 99.655 24.7 36.37 17 265.7179.5801 CAU.000580|USA. CAU.001139 2750 USA.AFR.000947 1 20.205 0 4.2853 43.41 13.0013 2751 USA.AFR.000558 1 8.735 5.555 10 6 34.58 10.35672752 SKE.AFR.000107 1 5.495 8.335 8.335 2 29.66 8.88317 2753USA.AFR.000440 1 5.495 8.335 8.335 2 29.66 8.88317 2754 EGY.AFR.000021 111.475 0 1.515 1 23.95 7.17302

Table 4 illustrates the example of scoring sample 3 against the mtDNAdatabase of 4441 entries (4840 original FBI mtDNA entries minus the 399sequences containing ambiguous base calls). The total combined score forthe HV1 and HV2 regions is shown in the column entitled “cumulativescore”. All entries are given a score relative to the highest cumulativescore in the column “% max score”. Database entry titles are in thecolumn “DB entries.” Sequences whose HV1 and HV2 PCR products areidentical are grouped into bins, with entry titles separated by verticallines. The cut-off point for this exercise was defined as 80% of the topcumulative score. The two bins that define this boundary are rows 47 and48. The total number of database entries that fall below this thresholdis 4347, or 97.9%.

Identification codes used in Table 4 are from the mtDNA populationdatabase (Miller K W Budowle B. Croat. Med. J 2001, 42(3), 315-27). AFR:African; CAU: Caucasian; ASN: Asian; CHN: Chinese; HIS: Hispanic; AUT:Austrian; EGY: Egypt; FRA: France; GRC: Greece; SKE: Sierra Leone.

Example 7 Optimization of Amplification Conditions and Reagents forEfficient Data Processing and Pattern Matching

Forensic analysis of human mtDNA by mass spectrometry presents a numberof challenges. First, PCR amplification reactions may result innon-templated additions of adenosine to the 3′-end of the template. Whenthis occurs, mass spectrum signals become mixed and detectionsensitivity is lowered. Second, the process of carrying out severalpurification steps to convert a PCR amplification mixture to appropriatespecific buffer conditions required for specific restriction digestsresults in significant sample loss. Lastly, a significant subset ofuseful restriction endonuclease enzymes yield double-stranded digestproducts with staggered ends. This occurrence has the effect ofcomplicating the process of restriction pattern analysis and limits thechoice of restriction endonucleases to those that only generateblunt-ended digestion products.

These complications have been solved by the use of exo(−) Pfu polymerase(Stratagene, La Jolla, Calif.), a 3′-5′ exonuclease-deleted Pfupolymerase. The mass spectra of FIG. 4 indicate that the use of exo(−)Pfu polymerase prevents the addition of non-templated adenosine residuesand 3′-end deletions which are normally observed when standard pfupolymerases are used. The resulting product exhibited a strong signal inthe mass spectrum. On the other hand, use of the commonly used Amplitaqgold polymerase (Applied Biosystems) did not circumvent this problem(FIG. 4). An additional advantage obtained through the use of exo(−) Pfupolymerase is that there is no need for purification of the PCR product.The PCR product mixture can be easily modified with appropriaterestriction enzyme activating buffer which is also compatible with theexo(−) Pfu polymerase.

A further additional advantage obtained from the avoidance of apurification procedure is that exo(−) Pfu polymerase remains viablethroughout the subsequent restriction digest process and this remainingpolymerase activity can be used to add leftover dNTPs to convertstaggered restriction products to blunt-ended products by filling in the“missing” nucleotide residues.

Thus, crude PCR products are directly subjected to the restrictiondigestion process, minimizing time, sample handling and potentialcontamination. FIG. 5 indicates that exo(−) Pfu polymerase is effectivefor consistent amplification of mtDNA obtained from blood, fingernailand saliva samples. PCR conditions for this experiment were as follows:A 50 μl reaction volume contained the following: 10 mM TRIS-HCl, 50 mMKCl, MgCl₂, 200 μM deoxynucleotide triphosphates, 400 mM betaine, 200 nMprimers, 4 units of Amplitaq Gold™ or 5 units exo(−) Pfu polymerase andmtDNA template and was subjected to incubation at 95° C. for 10 minutesfirst, then 35 cycles of the following thermal sequence: 95° C. for 20seconds, 52° C. for 20 seconds, 72° C. for 30 seconds. Following the 35cycles, the reaction was incubated at 72° C. for 4 minutes.

To take advantage of the modified function of the exo(−) Pfu polymerase,the experimental method was modified as follows: upon completion ofamplification of mtDNA, restriction endonucleases were added to theamplification mixture which was then incubated for 1 hour at 37° C. Thetemperature of the mixture was then raised to 37° C. for 15 minutes toactivate the exo(−) Pfu polymerase and enable the addition ofnucleotides to staggered ends to produce the blunt ends which facilitatepattern analysis.

As discussed above, the ability of exo(−) Pfu polymerase provides themeans of expanding the number of restriction endonucleases that arecompatible with the present method and simplifying data processing bysimplifying restriction digest patterns. Shown in FIG. 6 is the resultof a comparison of digest patterns obtained when the originally chosenrestriction enzymes EaeI and PacI are replaced with HaeIII and HpyCH4V.The pattern obtained using the newly chosen enzymes clearly results in arestriction digest pattern with better spacing of conserved restrictionsites which facilitates analysis. Shown in FIG. 7 is the result of a gelelectrophoresis analysis of the products of restriction digests. In thisexperiment a HV2 amplicon from a human mtDNA sample designated SeracareN31773. The mtDNA sample was amplified with Amplitaq Gold in 50 μlreaction volumes where 25 μl of PCR reaction was diluted up to 50 μl in:IX NEB restriction buffer #1, 10 mM Bis-TRIS Propane-HCl, 10 mM MgCl₂, 1mM DTT pH 7.0 (at 25° C.), 1×NEB BSA and (separately) 100 mg/μl in 1 μlvolumes of each enzyme as follows: EaeI: 3 units; HpyCH41V: 10 units;HpyCH4V: 5 units; HpaII: 10 units; PacI: 10 units; and HaeIII: 10 units.The mixtures were incubated for 1 hour at 37° C. before analysis in 4%agarose gel.

Restriction endonucleases MfeI and SspI are both useful alternatives toHpyCH4V and HpyCH41V respectively, because they cleave at similarpositions and cost significantly less than HpyCH4V and HpyCH41V.

Example 8 Validation of Mitochondrial DNA Analysis Method: Analysis ofHuman Cheek Swab mtDNA Samples and Comparison with the mtDNA PopulationDatabase

Cheek swabs were obtained from 16 volunteer donors. Genomic DNA wasisolated from the cheek swabs on a Qiagen MDx robot according toprocedures outlined in Example 1. Final elution volumes were 160 μl foreach well. 2 μl template was used in each PCR reaction which was runaccording to Example 1 except that the following cycling parameters wereused: 95° C. for 10 minutes followed by 45 cycles of 95° C. for 20 sec,52° C. for 20 sec and 72° C. for 30 sec, followed by holding at 72° C.for 4 minutes. Primer pairs used for HV1 were SEQ ID NOs: 12:15 and forHV2, SEQ ID NOs: 8:9.

PCR products (not shown) were digested with RsaI (HV1) or HaeIII, HpaII,HpyCH41V, and HpyCH4V (HV2) according to the procedure outlined inExample 2.

Restriction digests were performed in duplicate with each duplicateswab, followed by mass determination of the amplicon fragments by massspectrometry as described in Example 3. Samples were qualitativelyscored for HV1 and HV2 against each unique database entry by the sum of:

-   -   a) the percentage of expected fragments observed in the mass        spectrum;    -   b) the percentage of sequence positions covered by matched        masses; and    -   c) the total number of observed mass peaks accounted for by        matches to theoretical digest fragments.

Table 5 shows that, for the majority of the 16 samples, the ethnicdesignation of the majority of top-scoring entries from the FBI databasecoincide with the ethnic background of the donor. In general, mtDNAsequence data cannot be used to reliably associate a sample to theethnic background of the donor, because the mitochondria follow thematernal line exclusively and ethnic mixing in populations increases asthe general population becomes increasingly genetically integrated.However, as an overall assessment of the preliminary matching andscoring system, this association served well, because at the time ofthis evaluation, mtDNA samples had not been sequenced. Two outliers inthe association of donor ethnic background and major ethnic backgroundsof top database scores were samples 2 and 16. Sample 2 was anAfrican-American male with top database scores all designated“USA.CAU.xxx”. Upon inquiry, it was learned that this donor has aCaucasian mother. Because mtDNA is inherited maternally, the resultappears valid. TABLE 5 Results of Cheek Swab Comparison to the mtDNAPopulation Database Number % of % of % of % of Full of DB % of databasedatabase database database pattern entries database below below belowbelow match in with below 95% of 90% of 85% of 80% of Ethnicity mtDNAhighest highest highest highest highest highest closest Donor Donordatabase score score score score score score match Ethnicity 1USA.AFR.000975 1 99.979 99.979 99.959 99.917 99.917 AFR Af. Amer. 2USA.CAU.000191 3 99.938 99.731 99.153 96.054 92.169 CAU Af. Amer.USA.CAU.001303 With USA.CAU.001041 Cauc. Mother 3 None 1 99.979 99.93899.917 99.566 98.905 CHN Chinese 4 AUT.CAU.000080 22 99.545 98.12 96.77789.628 83.657 17 CAU Caucasian AUT.CAU.000090 4 HIS AUT.CAU.000099 2 AFRFRA.CAU.000041 18 more . . . 5 None 13 99.731 99.731 99.587 98.67897.417 12 CAU Caucasian 1 AFR 6 None 1 99.979 99.793 99.442 97.438 94.38CAU Caucasian 7 None 1 99.979 99.959 99.628 96.529 94.587 ASN Chinese 8None 1 99.979 99.876 99.793 98.244 96.157 CAU Caucasian 9 USA.CAU.0000311 99.979 99.979 99.979 99.256 98.574 CAU Caucasian 10 USA.CAU.000303 299.959 98.285 96.364 87.934 78.616 CAU Caucasian USA.CAU.000969 11 None2 99.959 99.959 99.835 99.814 99.36 ASN Chinese 12 USA.CAU.000113 199.979 99.897 99.07 98.099 92.149 CAU Caucasian 13 CHN.ASN.000374 1299.752 99.442 98.243 89.917 84.091 5 CAU Caucasian CHN.ASN.000411 3 ASNUSA.335.000122 3 AFR GRC.CAU.000007 1 335 9 others . . . 14USA.CAU.000297 1 99.979 99.979 99.917 99.649 95.806 CAU Caucasian 15None 1 99.979 99.959 98.037 92.417 86.59 ASN Indian (India) 16AUT.CAU.000096 12 99.752 99.669 98.863 95.971 84.7737 CAU IndianAUT.CAU.000100 (India) GRC.CAU.000011 USA.CAU.000604 4 others . . .

Identification codes used in Table 5 are from the mtDNA populationdatabase (Miller K W, Budowle B. Croat. Med. J 2001, 42(3), 315-27).AFR: African; CAU: Caucasian; ASN: Asian; CHN: Chinese; HIS: Hispanic;AUT: Austrian; GRC: Greece. Code 335 (USA. 335) in the donor 13 entryrefers to the U.S. territory of Guam.

Example 9: Expanding Discriminating Power of the Mitochondrial DNAAnalysis by Examination of Regions Outside of HV1 and HV2

Twelve regions of human mtDNA (referred to as R1-R12) were selected forinvestigation based upon a relatively large number of differencesbetween individual entries in 524 non-control-region human mitochondrialsequences obtained from Mitokor, Inc. (San Diego, Calif.). The initialtwelve primer pairs (see Table 3—SEQ ID NOs: 18:19, 20:21, 22:23, 26:27,28:29, 30:31, 32:33, 34:35, 36:37, 38:39, and 40:41) were tested upon˜1.6 ng of human blood-derived DNA (Seracare blood sample N31773) whichwas isolated as indicated in Example 1.

The PCR protocol and cycling conditions are as described in Example 1with the exception that 4 U of Amplitaq Gold polymerase (AppliedBiosystems, Foster City, Calif.) was used. The results of the reactionsare shown in FIG. 8 which indicates that reproducible amplicons wereobtained for all twelve non-control regions investigated.

Initial digestions with enzyme panels outlined in Example 2 wereemployed, and coverage maps were assembled by matching observed massesat +4 ppm error to all sequences existing in the database as of Sep. 8,2003-524 Mitokor-obtained sequences and 444 mtDNA genomes from GenBank.

The total number of unique sequences found within 968 predicted ampliconsequences from Mitokor and GenBank for each of the 12 non-control regionprimer pairs shows that the greatest number of different sequences isfound within regions R1, R3, R6, R7 and R9 (Table 6). When ampliconsequences are concatenated together as collinear sequences, thecombination of R1, R3, R6 and R7 comes out on top, with 508 unique basecount signatures out of 968 sequences predicted for the combinationR1+R3+R6+R7 compared to 475 unique signatures predicted for thecombination R1+R3+R9+R7. It was thus decided that regions R1, R3, R6 andR7 provide the best discriminating power. The numbers of uniquesequences for each of these regions are denoted by an asterisk in Table6. TABLE 6 Final Choices of Primers Optimized for Characterization ofNon-Control Mitochondrial DNA Regions RESTRICTION FORWARD REVERSE NO. OFUNIQUE NO. OF mtDNA REGION ENZYME SEQ ID SEQ ID BASE UNIQUE REGIONAMPLIFIED PANEL NO: NO: COMPOSITIONS SEQUENCES R1 COX2; DdeI MseI 18 49182  204* Intergenic HaeIII MboI spacer; tRNA- Lys; ATP6 R2 ND5 DdeIHaeIII 20 21 106 132 MboI MseI R3 ND6, tRNA-Glu; DdeI MseI 22 52 135 170* CYTB MboI BanI R4 COX3; tRNA-Gly; DdeI 24 25 94 132 ND3 HpyCH4IVMseI HaeIII R5 ND4L; ND4 AluI BfaI 26 27 107 130 MseI R6 CYTB; tRNA-Thr;DdeI HaeIII 57 29 118  143* tRNA-Pro MboI MseI RsaI R7 ND5; ND6 DdeIHpaII 58 31 137  174* HaeIII MseI R8 ND1 BfaI DdeI 32 33 88 122 EcoRIMboI R9 COX2; BfaI DdeI 34 35 118 145 Intergenic HpaII spacer; tRNA-HpyCH4IV Lys; ATP6 MboI  R10 COX1 BfaI HpaII 36 37 81 109 MboI  R11COX2; BfaI DdeI 38 39 113 136 Intergenic HpyCH4V spacer; tRNA- MboI Lys;ATP6  R12 16S rRNA; ND1 BfaI DdeI 40 43 65  79 MseI

The 12 regions were evaluated informatically by considering the totalnumber of unique sequences in each region out of a database of 968sequences, 524 of which were obtained from Mitokor, Inc, and 444 ofwhich are human mitochondrial genomes obtained from GenBank. Coordinatesare given in terms of the Anderson sequence (SEQ ID NO: 72). The numberof unique base count signatures was determined by theoretical digestionof each of the 968 database sequences with the indicated enzymes.

Example 10 Sensitivity Assessed With Quantified Human Blood DNA

To measure sensitivity against total human genomic DNA, a preparation ofDNA derived from whole human blood (Seracare blood sample N31774) wasobtained using the procedure of Example 1. A stock of blood-derived DNAwas quantitated to 1.6+0.06 ng/μl using the average of five independentconcentration measurements taken with the Molecular Probes PicoGreen®Assay P-7589. 10-fold serial dilutions of human DNA were tested in PCRreactions according to Example 1 using the primer pairs of SEQ ID NOs:12:15 (HV1) and SEQ ID NOs: 65:66 (HV2), starting with 1.6 ng/reactionand diluting to extinction (as a set of stock dilutions in doubledeionized H₂O) down to a calculated concentration of 160 zg/reaction (10orders of magnitude dilution). No carrier DNA was used in thesereactions.

FIG. 9 shows clear PCR product detection down to 1.6 pg/reaction forboth HV1 and HV2 primer pairs, with possible stochastic detection of afaint product at 160 fg input template. It is typically estimated that asingle human cell has approximately 3.3 billion base pairs-48, or 6.6billion total bases, which corresponds roughly to approximately 6-7 pgtotal DNA per cell. This suggests PCR detection of mtDNA targets down tosingle-cell or sub cellular levels.

After digesting HV1 amplicons with RsaI, and HV2 amplicons with HaeIII,HpaII, HpyCH41V and HpyCH4V, a full profile was recovered for HV2 with16 pg input template, and for HV1 with 160 pg input template. Subsequentexperiments have demonstrated full profile recovery for HV1 down to atabout 50 pg input template concentration with human DNA from the samesource. This represents an estimated 8 to 10 cells worth of DNA.

Example 11 Characterization of Mitochondrial DNA From Human Hair andSpecificity of HV1 and HV1 Primer Pairs in the Presence of Non-Human DNA

To test our ability to detect mitochondrial DNA from human hair shafts,and the specificity of our control-region primer targets in the presenceof non-human mammalian DNA, DNA was extracted from washed human hairshafts (8, 4, 2, 1 and 1 cm), washed hamster, dog, and cat hair (4-6 cm)and washed human (2-3 cm) plus hamster, dog or cat hair (4-6 cm) presenttogether in the same tube, according to the protocol outlined inExample 1. Hairs were taken by cutting with scissors, rather thanpulling to avoid including a hair root in the reactions. PCR reactionswere carried out using the primer pairs of SEQ ID NOs: 12:15 (HV1) andSEQ ID NOs: 65:66 (HV2) with PCR conditions as outlined in Example 1.Duplicate PCR reactions, demonstrated the presence of a PCR product ofthe expected size in the presence of human hair-derived DNA, but not inthe negative controls (identical reactions, but with double deionizedH₂O substituted for template) or with hamster, dog, or cat hair alone.

When these PCR were digested with RsaI (HV1) and HaeIII, HpaII,HpyCH41V, and HpyCH4V (HV2) as described in Example 2, a profile of basecompositions matching Ibis internal blinded sample CS0022 was found forproducts amplified in the presence of animal hair and for human hairalone down to 2 cm.

Example 12 Characterization of Mitochondrial DNA Isolated FourNon-Invasive Tissues (Cheek Swab, Hair, Fingernail and Saliva) fromThree Independent Donors: Analysis for Consistency in Processed MassSpectrometry Data

In this experiment, DNA was isolated from 3 pooled hairs of ˜2-3 cmlength each from 3 donors (designated “F”, “M” and “J”) according toprocedures outlined in Example 1. DNA from Several (3-5) pooled smallfingernail clippings was isolated from the same three donor according toExample 1 with the exception that there was no sonication step prior toDNA isolation, as this step was added at a later time. DNA from ˜0.5 mlsaliva was isolated from the same three donors according to Example 1.These three donors were also part of the 16-donor cheek swab paneldescribed in Example 8, and processed data from cheek swabs representingthese donors existed before this experiment and was used for comparisonto the three new tissue samplings.

PCR reactions were performed using 1 μl of template from each of thefour sample preparations for each of the three donors according toExample 1 using primer pair SEQ ID NOs: 12:15 (HV1) and SEQ ID NOs: 8:9(HV2). Restriction digestions were performed according to Example 2. Todetermine a truth base for each sample for this experiment, PCRreactions performed with primer pair SEQ ID NOs: 12:15 (HV1) and SEQ IDNOs: 8:9 (HV2) were purified with a QIAQuick PCR purification kit(according to Qiagen kit recommendations) and sequenced at Retrogen (SanDiego).

Digestion results for the original cheek swab-derived products werefirst compared to the sequences determined for cheek-swab-derivedamplicons for consistency. After confirming consistency between thedetermined sequence and the mass spectrometry derived fragment profile,the ability to qualitatively exclude each of the samples from the othertwo was evaluated by matching the processed mass data for the cheekswab-derived samples from each of the donors to theoretical digestionsfrom the PCR-derived sequences corresponding to the other two donors.

Processed mass spectrometry data for samples derived from the fourdifferent tissue sources were then compared to the cheek-swab-derivedsequence for each donor individually and found to be consistent acrossthe four tissue types, with the exception that the HV2 lengthheteroplasmy observed in HV2 of both sample “M” and sample “J” wasobserved in only three of the four tissue samples. The lengthheteroplasmy was not observed in the hair-derived sample for either “M”or “J”.

Example 13 Validation of Mitochondrial DNA Analysis Process on SalivaSamples from 36 Volunteer Donors

In this validation experiment, 1 μl of each of the 36 Ibis samples(CS0001-CS0036) was PCR amplified in duplicate using each of the finalprimer pairs shown in Table 6 on two different days (four reactions wereperformed on each sample) using the cycling parameters in indicated inExample 1. FIG. 10 shows one set of the 36 sample PCR reactions for theHV1 region. After PCR, 25 μl of each reaction were digested in 50 μlrestriction digestion reactions as described in Example 2. Samples fromeach of the 12 PCR plates were then subjected to mass spectrometry andprocessed with the ICR-2LS software to produce monoisotopic neutralmasses. Each set of mass data was scanned against the databaseindividually at +4 ppm matching threshold, allowing for the possibilityof a 1-dalton error on each mass determination.

One potential issue with the deconvolution from raw mass spectrometrydata to exact mass determination is the potential for the algorithm thatfits a theoretical isotopic distribution to an observed distribution canoccasionally predict the best fit with the distribution shifted byexactly one Dalton to the right or to the left of the true distribution,resulting in a mass determination that is exactly one Dalton off. Thisis not a serious issue when using mass data to verify consistency with aknown sequence, because the expected base composition is known and twoindependent measurements are made on each double stranded fragment whereeach strand (top and bottom) is linked to the other in a highlyconstrained manner because of base complementarity. When usingdeconvolved numerical masses to make de novo base compositionpredictions, however, this must be dealt with properly to ensure aproper interpretation of match data. For example, the mass differencebetween an internal ‘C’ and an internal ‘T’ in a DNA sequence is−14.9997 Daltons. The mass difference between an internal ‘G’ and aninternal ‘A’ is 15.9949 Daltons.

Because of this, the mass difference between two strands of DNA thatdiffer exactly by C T+G A is 0.9952 Daltons. Likewise, the reverse, TC+A G is a difference of −0.9952 Daltons.

For this reason, all of the matching to the database is performedassuming this as a possibility on every strand. However, when two massesmatch perfectly to two complementary base compositions at <10 ppm error(we generally use a threshold of 5 ppm or less) both masses wouldsimultaneously require a 1-dalton error, and both would be required tohave the error shifted in the same direction, to match a basecomposition fitting the above scenario. To avoid the rare occurrence ofthis situation, replicate reactions are required to ensure reproducibleresults for a profile analysis.

After scanning the database to generate a list of all possible fragmentmatches for each mass at +4 ppm threshold and allowing a precise+1Dalton error on every mass, an automatic filter was applied that assumesthat a pair of perfect matches to a complementary pair of basecompositions overrides a match requiring a 1-dalton shift in the samedirection on both strands (as described in the above paragraph). Asecond filter was applied to completely filter out ambiguous fragmentswhere one mass actually did exhibit a one-Dalton shift error. This iseasily spotted in an automated fashion, because two masses will onlymatch a complementary set of base compositions with high precision ifone of them is shifted by exactly 1 Dalton under this scenario. This canpresent ambiguity, however, because there is no de novo way to tellwhich mass has the error. Replicate reactions are relied upon to resolvethis type of ambiguity (alternatively, a profile can be scanned withambiguity in an “either-or” mode with little or no effect on the actualmatch result if enough fragments are present in a profile, much likeusing an ‘R’ to represent ‘A’ or ‘G’, or an ‘N’ to represent anynucleotide).

The last step is to create a composite profile from the combination ofpre-filtered matches in each reaction scenario. To do this, all of theunfiltered masses from each of the replicates in each reaction scenario(e.g., one reaction scenario would be HV1 PCR product digested withRsaI) were combined into one data set and used again against the entiredatabase to regenerate a single composite profile. This operationprovides the benefit of increasing sensitivity in that a fragment lostin one reaction can be picked up in another, and can help preventambiguous base composition assignments. The final step is to filter anyambiguous assignments from the composite profile before comparingprofiles or scanning the database with a profile. Even in the veryunlikely case that masses representing both strands of a fragment wereDalton-shifted in the same direction, the same fragment in a replicatereaction should disagree, which is the precautionary purpose of thefinal filtering step.

Table 7 summarizes the results of the database scans using thesix-region profiles. It should be noted here that there was considerablymore noise in the larger non-control-region spectra than the spectra forthe HV1 and HV2 regions. Although it did not detract from the ability tomatch the proper donor signature, it did produce more than desiredambiguity in data processing. The level of noise in this data set alsodid not cause a problem in the ability to differentiate samples fromeach other by at least one SNP, with the exception of samples CS0004,CS0025 and CS0032. Interestingly, one SNP in R1 differentiates CS0004and CS0025 (which appear to a very common mtDNA type when HV1 and HV2are matched to the database), which was detected only in the CS0025profile. Therefore, CS0004 and CS0025 could not be resolved from eachother by direct comparison (see next section), CS0004 hits equally toCS0004 and CS0025 in the database scan, and CS0025 appears todifferentiate from CS0004 in a database scan (due to the fact that theprofile is being compared to the known CS0004 sequence in the lattercase, rather than the experimentally determined base composition profilethat has a missing fragment). Two incorrect base compositions werepredicted in CS0018 that were corrected by analysis of a duplicate setof restriction digestions. One incorrect base composition was predictedin each of samples CS0006, CS0011 and CS0026, each of which was likewisecorrected by analysis of a duplicate set of restriction digestions. Thisdid not change the top database hit (Table 7), nor does it change theability of CS0001—CS0036 to be differentiated from CS0018, CS0006,CS0011, or CS0026. TABLE 7 Overview of Validation Results % NO. OF NO.OF MATCHED SECOND BEST % NO. OF ID WITH BEST DATABASE FRAGMENTS MATCHINGREFERENCE FRAGMENTS SECOND BEST SAMPLE MATCH MATCH HIGHEST % POSITIONSMATCHED FRAGMENTS MATCHED CS0001 CS0001 100 1 2942 90 2 CS0002 CS0002100 1 3356 95.3 2 CS0003 CS0003 100 1 3294 90.7 2 CS0004 CS0004 100 62879 97.3 14 CS0025 CS0032 gi|17985669 gi|13272808 gi|7985543 CS0005CS0005 100 1 3190 95.1 12 CS0006 CS0006 97.5 1 3088 92.5 2 CS0006 CS0006100 1 3198 95 2 Re-anal. CS0007 CS0007 100 1 2940 87.2 11 CS0008 CS0008100 1 3251 95.2 2 CS0009 CS0009 100 1 2617 89.2 6 CS0010 CS0010 100 23205 97.6 8 gi|32692659 CS0011 CS0011 97.7 1 3086 90.7 5 CS0011 CS0011100 1 3028 92.7 5 Re-anal. CS0012 CS0012 100 1 3193 92.7 2 CS0013 CS0013100 1 3016 87.5 4 CS0014 CS0014 100 1 3017 92.5 1 CS0015 CS0015 100 13378 95.3 1 CS0016 CS0016 100 1 2915 94.7 1 CS0017 CS0017 100 1 322992.9 3 CS0018 CS0018 94.9 1 2629 89.7 1 CS0018 CS0018 100 1 2691 94.4 1Re-anal. CS0019 CS0019 100 1 2794 92.3 1 CS0020 CS0020 100 1 3231 92.9 8CS0021 CS0021 100 1 2902 97.5 3 CS0022 CS0022 100 1 3314 95.3 10 CS0023CS0023 100 1 2953 84.6 3 CS0024 CS0024 100 1 3224 87.8 1 CS0025 CS0025100 3 3080 97.6 11 gi|3272808 gi|7985669 CS0026 CS0026 97.6 1 2787 90.21 CS0026 CS0026 100 2787 92.5 1 Re-anal. CS0027 CS0027 100 1 2940 94.9 4CS0028 CS0028 100 1 2975 97.5 8 CS0029 CS0029 100 1 3002 92.7 4 CS0030CS0030 100 1 3066 97.6 1 CS0031 CS0031 100 1 3409 86.4 7 CS0032 CS0032100 2 3288 97.6 3 gi|17985543 CS0033 CS0033 100 1 3098 92.7 2 CS0034CS0034 100 1 3100 85.4 2 CS0035 CS0035 100 3 2971 97.5 3 gi|32892351gi|32892449 CS0036 CS0036 100 1 2703 91.9 3

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference cited in the presentapplication is incorporated herein by reference in its entirety.

1. A method of forensic analysis of a sample comprising mitochondrialDNA comprising: selecting a region of mitochondrial DNA comprising atleast one restriction site whereat a restriction enzyme cleaves saidmitochondrial DNA to produce a plurality of restriction fragments;populating a relational database of known mitochondrial DNA sequenceswith molecular masses which correspond to theoretical restrictionfragments obtained from theoretical digestion of each member of saiddatabase at said at least one restriction site; selecting a primer pairwith which to amplify said region of mitochondrial DNA in said sample;amplifying said region of mitochondrial DNA in said sample to produce anamplification product; digesting said amplification product with atleast one restriction enzyme to produce a plurality of restrictionfragments; experimentally determining the molecular masses of eachmember of said plurality of restriction fragments; and comparing saidexperimentally determined molecular masses with the molecular masses ofsaid theoretical digestion of each member of said database, wherein atleast one match or lack of a match provides a forensic conclusion. 2.The method of claim 1 wherein said region of mitochondrial DNA comprisesHV1.
 3. The method of claim 2 wherein each member of said primer pairhas at least 70% sequence identity with the sequence of thecorresponding member of any one of the following primer pair sequences:SEQ ID NOs: 12:13, 12:14, 12:15, 16: 17, 42:43, 42:46, 67:68, 69:70,12:68, 12:70, 67:15, 71:70, 69:15 and 69:68.
 4. The method of claim 2wherein said at least one restriction enzyme is RsaI.
 5. The method ofclaim 1 wherein said region of mitochondrial DNA comprises HV2.
 6. Themethod of claim 5 wherein each member of said primer pair has at least70% sequence identity with the sequence of the corresponding member ofany one of the following primer pair sequences: SEQ ID NOs: 8:9, 10:11,16:17 and 65:66.
 7. The method of claim 5 wherein said at least onerestriction enzyme is HaeIII, HpaII, MfeI, or SspI.
 8. The method ofclaim 5 wherein said at least one restriction enzyme is HpaII, HpyCH4IV,PacI, or EaeI.
 9. The method of claim 1 wherein said region ofmitochondrial DNA comprises region R1.
 10. The method of claim 9 whereineach member of said primer pair has at least 70% sequence identity withthe sequence of the corresponding member of any one of the followingprimer pair sequences: SEQ ID NOs: 18:19 and 18:49.
 11. The method ofclaim 9 wherein said at least one restriction enzyme is DdeI, MseI,HaeIII, or MboI.
 12. The method of claim 1 wherein said region ofmitochondrial DNA comprises region R2.
 13. The method of claim 12wherein each member of said primer pair has at least 70% sequenceidentity with the sequence of the corresponding member of any one of thefollowing primer pair sequences: SEQ ID NOs: 20:21 and 50:51.
 14. Themethod of claim 12 wherein said at least one restriction enzyme is DdeI,HaeIII, MboI, or MseI.
 15. The method of claim 1 wherein said region ofmitochondrial DNA comprises region R3.
 16. The method of claim 15wherein each member of said primer pair has at least 70% sequenceidentity with the sequence of the corresponding member of any one of thefollowing primer pair sequences: SEQ ID NOs: 22:23 and 22:52.
 17. Themethod of claim 15 wherein said at least one restriction enzyme is DdeI,MseI, MboI, or BanI.
 18. The method of claim 1 wherein said region ofmitochondrial DNA comprises region R4.
 19. The method of claim 18wherein each member of said primer pair has at least 70% sequenceidentity with the sequence of the corresponding member of any one of thefollowing primer pair sequences: SEQ ID NOs: 24:25 and 53:54.
 20. Themethod of claim 18 wherein said at least one restriction enzyme is DdeI,HpyCH4IV, MseI, or HaeIII.
 21. The method of claim 1 wherein said regionof mitochondrial DNA comprises region R5.
 22. The method of claim 21wherein each member of said primer pair has at least 70% sequenceidentity with the sequence of the corresponding member of any one of thefollowing primer pair sequences: SEQ ID NOs: 26:27 and 55:56.
 23. Themethod of claim 21 wherein said at least one restriction enzyme is AluI,BfaI, or MseI.
 24. The method of claim 1 wherein said region ofmitochondrial DNA comprises region R6.
 25. The method of claim 24wherein each member of said primer pair has at least 70% sequenceidentity with the sequence of the corresponding member of any one of thefollowing primer pair sequences: SEQ ID NOs: 28:29 and 57:29.
 26. Themethod of claim 24 wherein said at least one restriction enzyme is DdeI,HaeIII, MboI, MseI, or RsaI.
 27. The method of claim 1 wherein saidregion of mitochondrial DNA comprises region R7.
 28. The method of claim27 wherein each member of said primer pair has at least 70% sequenceidentity with the sequence of the corresponding member of any one of thefollowing primer pair sequences: SEQ ID NOs: 30:31 and 58:31.
 29. Themethod of claim 27 wherein said at least one restriction enzyme is DdeI,HpaII, HaeIII, or MseI.
 30. The method of claim 1 wherein said region ofmitochondrial DNA comprises region R8.
 31. The method of claim 30wherein each member of said primer pair has at least 70% sequenceidentity with the sequence of the corresponding member of any one of thefollowing primer pair sequences: SEQ ID NOs: 32:33 and 59:60.
 32. Themethod of claim 30 wherein said at least one restriction enzyme is BfaI,DdeI, EcoRI, or MboI.
 33. The method of claim 1 wherein said region ofmitochondrial DNA comprises region R9.
 34. The method of claim 33wherein each member of said primer pair has at least 70% sequenceidentity with the sequence of the corresponding member of the followingprimer pair sequences: SEQ ID NOs: 34:35.
 35. The method of claim 33wherein said at least one restriction enzyme is BfaI, DdeI, HpaII,HpyCH4IV, or MboI.
 36. The method of claim 1 wherein said region ofmitochondrial DNA comprises region R10.
 37. The method of claim 36wherein each member of said primer pair has at least 70% sequenceidentity with the sequence of the corresponding member of the followingprimer pair sequences: SEQ ID NOs: 34:35.
 38. The method of claim 36wherein said at least one restriction enzyme is BfaI, HpaII, or MboI.39. The method of claim 1 wherein said region of mitochondrial DNAcomprises region R10.
 40. The method of claim 39 wherein each member ofsaid primer pair has at least 70% sequence identity with the sequence ofthe corresponding member of the following primer pair sequences: SEQ IDNOs: 36:37 and 61:62.
 41. The method of claim 39 wherein said at leastone restriction enzyme is BfaI, HpaII, or MboI.
 42. The method of claim1 wherein said region of mitochondrial DNA comprises region R11.
 43. Themethod of claim 42 wherein each member of said primer pair has at least70% sequence identity with the sequence of the corresponding member ofthe following primer pair sequences: SEQ ID NOs: 38:39 and 63:39. 44.The method of claim 42 wherein said at least one restriction enzyme isBfaI, DdeI, HpyCH4V, or MboI.
 45. The method of claim 1 wherein saidregion of mitochondrial DNA comprises region R12.
 46. The method ofclaim 45 wherein each member of said primer pair has at least 70%sequence identity with the sequence of the corresponding member of thefollowing primer pair sequences: SEQ ID NOs: 40:41 and 40:64.
 47. Themethod of claim 45 wherein said at least one restriction enzyme is BfaI,DdeI, or MseI.
 48. The method of claim 1 further comprising: populatingsaid relational database of known mitochondrial DNA sequences with basecompositions which correspond to theoretical restriction fragmentsobtained from theoretical digestion of each member of said database atsaid at least one restriction site; experimentally determining the basecomposition of each member of said plurality of restriction fragmentsfrom said experimentally determined molecular masses of each member ofsaid plurality of restriction fragments; comparing said experimentallydetermined base compositions with the base compositions of saidtheoretical digestion of each member of said database wherein at leastone match or lack of a match provides a forensic conclusion.
 49. Themethod of claim 1 wherein said amplifying step comprises polymerasechain reaction.
 50. The method of claim 49 wherein said polymerase chainreaction is catalyzed by a polymerase enzyme whose function is modifiedrelative to a native polymerase.
 51. The method of claim 50 wherein saidmodified polymerase enzyme is exo(−) Pfu polymerase.
 52. The method ofclaim 50 wherein said modified polymerase catalyzes the addition ofnucleotide residues to staggered restriction digest products to convertsaid staggered digest products to blunt-ended digest products.
 53. Themethod of claim 1 wherein said amplifying step comprises ligase chainreaction or strand displacement amplification.
 54. The method of claim 1wherein said database is a human mtDNA population database.
 55. Themethod of claim 1 wherein said molecular masses are determined byESI-FTICR mass spectrometry.
 56. The method of claim 1 wherein saidmolecular masses are determined by ESI-TOF mass spectrometry.
 57. Themethod of claim 1 further comprising repeating all steps of the methodfor at least one additional region of mitochondrial DNA.
 58. The methodof claim 57 wherein said at least one additional region is from HV1,HV2, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, or R12.
 59. Themethod of claim 1 wherein said mitochondrial DNA is human mitochondrialDNA.
 60. The method of claim 1 wherein said mitochondrial DNA is animalmitochondrial DNA.
 61. The method of claim 1 wherein said mitochondrialDNA is fungal, parasitic, or protozoan DNA.
 62. The method of claim 1wherein said amplified DNA is digested directly without purification.63. The method of claim 1 wherein said sample of mitochondrial DNA isobtained from saliva, hair, blood, or nail.
 64. The method of claim 1wherein said plurality of restriction fragments are up to about 150 basepairs in length.
 65. A primer pair wherein each member of the pair hasat least 70% sequence identity with the sequence of the correspondingmember of any one of the following primer pair sequences: SEQ ID NOs:8:9, 10:11, 12:13, 12:14, 12:15, 16:17, 18:19, 20:21, 22:23, 24:25,26:27, 28:29, 30:31, 32:33, 34:35, 36:37, 38:39, 40:41, 42:43, 44:45,42:46, 47:48, 18:49, 50:51, 22:52, 53:54, 55:56, 57:29, 58:31, 59:60,61:62, 63:39, 40:64, 65:66, 67:68, 69:70, 12:68, 12:70, 67:15, 71:70,69:15, and 69:68.
 66. The primer pair of claim 65 comprising at leastone modified nucleobase.
 67. The primer pair of claim 66 wherein themodified nucleobase is 5-propynylcytidine or 5-propynyluridine.
 68. Anisolated mitochondrial DNA amplicon comprising a segment ofmitochondrial DNA produced by the process of amplification of a sampleof mitochondrial DNA with the pair of primers of claim 65.